Characterisation of ACP5 missense mutations encoding tartrate-resistant acid phosphatase associated with spondyloenchondrodysplasia.

Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.

Introduction

Tartrate-resistant acid phosphatase (type-5 acid phosphatase; TRACP, EC 3.1.3.2.) is a ~35 kD metalloenzyme with a mixed valency di-iron center, required for catalytic activity [1]. TRACP is expressed primarily by differentiated cells of the mononuclear phagocyte system including osteoclasts, macrophages and dendritic cells (DC) [2]. It has a long history of clinical relevance as a cytochemical marker for hairy cell leukaemia, and as a serum marker for osteoclastic bone resorption and, more recently, chronic inflammation [3]. TRACP functions in vitro cleaving unnatural phosphates like p-nitro-phenyl phosphate but its biological role in bone resorption and immune responses may be different [1]. One natural substrate is osteopontin (1). TRACP exists as two isoforms, which are derived by differential post-translational processing of a central regulatory loop peptide [4]. TRACP5a, a monomeric protein, with the intact loop peptide, has a lower pH optimum of ~5 and specific activity of ~100 U/mg. TRACP5b is proteolytically cleaved into a 23 kD and 16 kD disulphide linked heterodimer with a pH optimum of ~6 and specific activity of ~1000 U/mg [5]. In addition, isoforms 5a and 5b are differentially compartmentalized. In macrophages and dendritic cells, only isoform 5a is secreted from cells; isoform 5b remains intracellularly [6]. Therefore, serum TRACP5a serves as a marker for systemic macrophage functions and chronic inflammatory activity [3]. In osteoclasts, however, isoform 5b is released into the circulation with other matrix products during bone resorption, thus serving as a marker for osteoclast activity [6]. The regulatory signals that govern this differential processing are not fully understood.

The complex inherited disease spondyloenchondrodysplasia (SPENCD) is a recently described disorder comprising of craniofacial, skeletal, neurological and autoimmune manifestations [7, 8, 9]. More specifically, these include skeletal dysplasia and radiolucent metaphysical lesions which arise from biallelic mutations of ACP5 gene, which encodes TRACP enzyme [9-11]. We and others have shown earlier [12, 13, 14] that mutations of ACP5 can cause autoimmune cytopenia, immuno-osseous dysplasia, spasticity with leukodystrophy, systemic lupus erythematosus (SLE), Moyamoya syndrome and Sjogren’s syndrome [15-17]. Other salient features of SPENCD include retardation of growth with developmental delays, clumsy movements and specific neurological symptoms such as intracranial/cerebral calcifications. Increased expression of type-I INF regulated genes which tracks parallel with extra skeletal abnormalities has also been observed [12-14].

ACP5 is transcribed from a single gene with 5 exons. Three alternate promoters exist within the first three exons (E1A, E1B and E1C) [18]. The TRACP protein is translated from exons 2 to 5. Molecular studies such as promoter analysis and assignment of chromosomal localizations have been under taken by Reddy et al., [19] for human and mouse genes. Molecular modelling of the eight-missense mutant TRACP proteins associated with SPENCD suggested that single amino acid substitutions could lead to protein destabilization [12-14]. In SPENCD, ACP5 consists of partial or whole gene deletions and nonsense or missense single base substitutions. Seventeen distinct mutations have so far been reported by two groups including ours (4 deletions, 5 nonsense mutations and 8 missense single base changes) [9, 10]. Of the ten patients in whom TRACP activity or protein was studied, no detectable TRACP activity was lacking in cells (4 patients) or no circulating TRACP protein was found in serum (6 patients) (unpublished). The clinical presentation reinforces the concept that TRACP is a member of the growing number of molecules important in osteoimmunology, and may be a key pathophysiological player. It may also be a therapeutic target in metabolic bone diseases [20], immune disorders [21, 22] and cancer [23, 24].

The overall purpose of the study is to clone and express all the missense ACP5 genes in human embryonic kidney-293 (HEK-293) cells which are analogous to those observed with SPENCD related mutations in humans [12-14]. The resultant TRACP protein products were characterized in human cell lines to define the effects of the specific amino acid changes and provide direct evidence for the causal mechanism of TRACP deficiency in SPENCD patients. From a practical perspective, these clinically relevant mutations were exploited in human derived stable cell lines to learn more about the specific epitopes targeted by unique monoclonal antibodies to TRACP enzyme developed in our laboratory.

Materials and methods

Transformation of mutant genes and subcloning

Wild type and eight missense ACP5 mutant genes [12, 13] were synthesized and cloned directly into a proprietary pJ602 vector containing the cytomegalovirus (CMV) promoter using ampicillin and Zeocin resistance genes for selection (ATUM formally DNA 2.0, Menlo Park, CA). ACP5 genes contained single base changes leading to amino acid substitutions: K52T (c.155A>C), T89I (c.266C>T), G109R (c.325G>A), L201P (c.602T>C), G215R (c.643G>A), D241N (c.721G>A), N262H (c.784A>C), and M264K (c.791T>A) (). Single colonies of transformed E. coli were isolated on LB plates containing 100 μg/ml of ampicillin (LB-amp). Plasmid DNAs were prepared from 500 ml of LB-amp broth cultures using Maxi-prep kits (Qiagen Inc, Valencia, CA). Human monocyte derived and recombinant TRACP proteins were prepared as described earlier [25, 26].

Amino acid sequence of human TRACP.

Amino acid substitutions due to the observed missense mutations are shown below the wild (WT) sequence. Functionally important regions, including the leader peptide and regulatory loop peptides are underlined. Iron-binding two domains are represented in bold italic. N-linked glycan acceptor asparagines are noted by an asterisk (*) on the top of the residue.

Transient transfection and generation of stable cell lines

HEK-293 cells were transfected with one to two μg of plasmid DNAs using Fugene 6 reagent (Roche Applied Science, Indianapolis, IN) according to recommended procedures. Cells were screened for TRACP expression after 48 hours, after which cytochemical and immunocytochemical techniques [27, 28] were carried out as described below. Transiently transfected cells and their supernatant media were harvested after 72 hours. Cytocentrifuge smears were prepared from the cells, air dried and stored at room temperature until used for staining. Media were stored in aliquots at –70°C until used for analyses. The cell lysates were prepared at 107cells/ml in a buffer of 10 mM Tris (hydroxymethyl) aminomethane (TRIS), 1 mM Ethylene Diamine Tetraacetate (EDTA), 1 mM Ethylene Glycol-bis-(aminoethyl ether) Tetraacetate (EGTA), 1% Nonidet P-40 (NP-40), 300 mM NaCl and protease inhibitor cocktail (2 mM phenyl methyl sulfonyl fluoride (PMSF) / 10 μg/ml aprotinin / 10 μg/ml leupeptin, at pH 7.4). After 30 minutes on ice, the lysates were cleared of insoluble debris by centrifugation. Total protein in cell lysates was determined by a commercial Coomassie Blue dye binding method (Bio-Rad, Richmond, CA) using bovine serum albumin (BSA) as standard. For all immunoassays and immunoblots, the cellular protein input for mutant TRACP was normalized to that of wild type (WT) TRACP-expressing cells (4 μg/μl). Soluble proteins were stored at –70°C until used for analyses. Stable transfectants were selected using 10 to 100 μg Zeocin/ml. After 7–14 days, growing colonies were picked and expanded in the presence of Zeocin and re-picked until the growing clones were 100% positive for TRACP expression by immuno-cytochemical staining. Stable clones are grown in the continuous presence of 10–100 μg Zeocin/ml. Media and cell lysates were also prepared from the stable transfectant clones.

Control dendritic cell preparations

Dendritic cells (DC) were differentiated from blood monocytes as described earlier [25]. Monocytes were isolated by density gradient centrifugation of buffy coat cells from a de-identified therapeutic phlebotomy specimen from a patient with hemochromatosis. Enriched monocytes were cultured in 10 cm dishes at 106 cells/ ml in RPMI-1640 medium supplemented with antibiotics, 10% fetal bovine serum, 25 ng/ml GM-CSF and 20 ng/ml interleukin 4. An additional dose of cytokines was added at day four. On day six, non-adherent cells were decanted, and the dishes were rinsed gently once with Hank’s balanced salt solution (HBSS). Five ml of HBSS containing 2 mM EDTA was added to detach the differentiated DC. Cells were washed 3 times in HBSS, pelleted and lysed at 107 cells/ml and the protein content was determined as described above. TRACP secreted to medium was enriched by one-step purification by ion exchange chromatography [18, 19]. One hundred-twenty ml of DC culture medium after day six was collected and adjusted to pH 5.0, clarified by centrifugation and applied to a 5 ml column of SP Sepharose (GE Healthcare). After washing the column with washing buffer (10 mM Na acetate/50 mM NaCl), the bound TRACP was eluted with elution buffer 10 mM Na acetate/1.0 M NaCl, and ten 1.0 ml fractions were collected. TRACP activity was determined using 10 μl of each fraction in 200 μl of 10 mM, 4-Nitrophenyl phosphate in 100 mM Na acetate/50 mM sodium tartrate buffer, pH 5.5. The active peak fractions were pooled and dialyzed against 50 mM Na acetate buffer pH 5.5 containing 100 mM NaCl/2% glycerol.

TRACP cytochemistry and immunocytochemistry

Cytocentrifuge smears were prepared and stained for TRACP activity using naphthol ASBI-phosphate as substrate and hexazotized pararosaniline as coupler at pH 5.5 according to published methods [27]. TRACP proteins were stained immuno-cytochemically using monoclonal antibody, Mab220, for intact TRACP5a like isoform, Mab9C5 for total TRACP protein and 5b isoform as well [28]. Control smears were also stained using IgG from non-immune ascites. Fixed smears were subjected to heat-induced epitope retrieval for 30 minutes at 70°C in a commercial solution of EDTA at pH 8.0 (Invitrogen Corp, Carlsbad, CA), followed by 15 seconds permeabilization in 0.2% Triton X-100/PBS prior to immunochemical staining.

Immunoassay for TRACP activity and protein

Antibodies used for immunoassays were all developed in our laboratory [25-28]. Monoclonal antibodies, 14G6 (Mab14G6) and Mab162 target independent conformational epitopes on both isoforms 5a and 5b and are used to measure TRACP5b activity and total TRACP protein in native conformation. These epitopes are destroyed by heating in sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. Mab220 targets the trypsin-sensitive regulatory loop peptide present only in isoform 5a and reacts with both native and denatured forms of the protein. Mab220 is used as capture antibody to measure isoform 5a activity and protein specifically. Mab9C5 targets a linear epitope in the C-terminal part of both isoforms 5a and 5b after heat denaturation. It is suitable for Western blot analysis of both TRACP5a (35 kD) and 5b (16 kD) but does not react with native enzyme. Mab9C5 is used as capture antibody to measure denatured or non-native precursor TRACP protein [25, 26].

TRACP isoform activities were measured in growth media, and cell lysates according to our published methods [25, 26]. After immobilization of TRACP with Mab220 (isoform 5a) or Mab14G6 (total TRACP), the bound enzyme activity was determined using 4-nitrophenyl phosphate at pH 5.8 and 6.1 respectively. Assays were standardized against a serial dilution of 4-nitrophenolate equivalent to 0.16 to 10 IU (μmol 4-Nitrophenyl phosphate (4-NPP) hydrolysed/minute/liter). TRACP isoform proteins were immobilized with Mab220 (native isoform 5a), Mab14G6 (total native TRACP) or Mab9C5 (total denatured TRACP including 5b isoform). Immobilized native TRACP proteins were detected with a unique anti-TRACP Mab162 conjugated to horseradish peroxidase (Mab162-HRP). Immobilized denatured TRACP protein was detected with Mab220-HRP. Peroxidase activity was determined with o-Phenylene diamine dihydrochloride and H2O2 [25, 26]. Native TRACP protein assays were estimated using serial dilutions of partially pure serum TRACP5a (0.08 to 5 ng/ml). Results for the denatured TRACP assay are expressed as A490 absorbance values.

Western blotting

To determine the effects of TRACP mutations the expressed proteins from culture medium (10 μl) and cell lysates (107 cells/ml) were subjected to Western (immuno) blot analysis using Mab220 (TRACP5a) and Mab9C5 (TRACP 5a and 5b) as probes [28]. Ten μl medium was used for analysis of all cell culture medium samples. Protein amounts in lysates were normalized to that in 2 μl of WT. Samples were heated to 100°C for 2 minutes under reducing conditions using 2-mercapto ethanol, and electrophoresed in 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis proteins were transferred onto polyvinylidene fluoride (PVDF) membranes for immuno-detection [29]. The membranes were blocked with 3% milk powder for 1 h at room temperature in 1% Tween 20 in TBS (20 mM Tris, pH 7.5, 0.5 M NaCl) and washed three times in TBST (TBS + 0.05% Tween 20). Primary antibodies were used in TBST as well and washed again for three times followed by secondary antibody conjugated with alkaline phosphatase (rabbit anti-mouse IgG). Immuno blot color development was carried out with Nitro Blue Tetrazolium chloride/ 5-bromo-4-chloro-indol-3-yl phosphate p-toluidine salt following the modified method of Brenan and Bath [30] based upon original methods of Van Noorden [31] and McGadey [32]. Blots finally washed with water, fixed in EDTA and dried at room temperature.

Deglycosylation and proteolysis of TRACP proteins

To determine if mutations had cause altered post-translational modifications, cell lysates and supernatant media were exposed to endoglycosidase-H (Endo-H; Boehringer Mannheim Biochemicals, Indianapolis, IN) or peptide N-glycosidase F (PNGaseF; New England Biolabs, Beverly, MA) after mild denaturation. Amounts normalized to protein in 2 μl WT cell lysate (4 μg/μl) or 10 μl culture medium were mixed in triplicate with 1 μl PNGase F denaturing buffer (NEB) and water was added to make 10 μl. Samples were boiled for 10 minutes to denature protein and expose oligosaccharides. Then 0.5 mU (1 μl) Endo-H and 1 μl 10X buffer (500 mM Na citrate / 10% NP-40, pH 5.5), or 500U (1 μl) PNGase F and 1 μl 10 X buffer (500 mM NaPO4 / 10% NP-40, pH 7.0), or 1 μl 10X Endo H buffer were added to the denatured samples. Digestion was carried out at 37°C for 60 minutes. An equal volume of SDS-PAGE reducing sample buffer was added and the samples were stored at –70°C until used for Western blot analysis with Mab9C5.

Limited proteolysis of TRACP proteins with trypsin

To examine if mutant intracellular TRACPs could be activated by proteolysis in vitro from isoform 5a-like to 5b-like enzymes, TRACP-HEK lysates in 2 μl WT cells or 10 μl culture medium were digested with 200 mU solid-phase trypsin-agarose (Sigma Chem Co, St. Louis, MO) or control BSA-conjugated agarose (2 mg BSA /ml packed agarose) made with Amino Link Plus coupling gel (Pierce Chemical Co., Rockford, IL) for 2h at room temperature. Reactions were made to 50 μl with 10 mM Tris / 30 mM NaCl / 0.1% Triton X-100, pH 7.0. After incubation, the solid-phase trypsin and BSA were removed by centrifugation. Digests were subjected to Western blot analysis with Mab9C5.

Statistical analysis

All data were analysed using GraphPad Prism Version 8.0.1. For all measurements, student T-test was used to assess the statistical significance between groups. All experiments were carried out at least three times before arriving at statistical differences. Statistically significant difference was as follows: **** highly significant (p<0.00005), *** moderately significant (p<0.0005), ** significant (p<0.005), * less significant (p<0.05).

Results

Amino acid sequences of TRACP holo enzyme and its structural features

illustrates the entire amino acid sequence of human holo-TRACP with leader and loop peptides. Missense mutations observed in SPENCD are depicted below the wild (WT) sequences. In the homozygous or heterozygous state, these mutations result in TRACP deficiency in cells and serum resulting in a complex heritable disease SPENCD [12-14]. Apart from the leader peptide (1–21 residues), when the loop peptide is removed it increases the enzyme activity converting TRACP 5a into 5b isoform. In the loop peptide resides between 162–182 residues. The iron binding domains of TRACP mediating ROS generation are indicated by bold italics in which lie between residues 105–113 and 240–247. Two distinct N-glycosidic linkage acceptor asparagines (116 and 147) exist in TRACP as indicated. This type I linkage with the canonical amino acid triplets NVS (; amino acids 116–118 and147-149) are well conserved and destined to classical ER mediated processing of linked carbohydrate moieties.

Recombinant mutant TRACP expression

Transient (72 hr) and stable expression of mutant TRACP proteins were confirmed by cytochemical staining for TRACP activity. Immuno-cytochemical stainings were carried out using Mab220 (TRACP5a) and Mab9C5 (total TRACP). As shown in WT TRACP only expresses strong activity while all mutants lacked stainable activity. However, immuno-cytochemical staining for intact TRACP protein (Mab220) showed that expression was similar in all transient transfectants and that stain intensity was strong for all mutants almost equivalent to that of WT TRACP. Immuno-staining with Mab9C5 demonstrated similar results analogous to those with Mab220 (not shown). These results indicate no gross inhibition of TRACP polypeptide biosynthesis by the mutations; but the mutations likely interfere with enzymatic function(s).

Cytocentrifuge smears were prepared from WT and mutant TRACP cells at 72 hours after transfection.

Smears were fixed and stained directly for TRACP enzyme activity (A to I) or immuno-cytochemical staining for intact TRACP protein with Mab220 (J to R) was carried out as a probe. Only wild (WT) TRACP was active, all mutants cells expressed as inactive proteins. Experiments were repeated with five slides at a time and the best stained pictures were presented.

Expression of intracellular and secreted recombinant TRACP proteins

In contrast to the immuno-cytochemical results, quantitative immunoassay for TRACP isoforms secreted into medium () and expressed intracellularly ( for TRACP activity 5a and 5b respectively; for total protein) initially showed an almost complete lack of TRACP expression in six out of eight missense mutations examined. Only T89I and M264K mutants were detectable to an appreciable degree as inactive proteins (). This is explained by the fact that Mab14G6 used to capture total TRACP and Mab162-HRP used as detection antibody in immunoassays react with only the native conformation of TRACP [33]. Therefore, the T89I and M264K mutations likely result in the synthesis of enzymes with at least partial native conformations conserved at the Mab14G6 and Mab162 epitopes, in addition to the Mab220 loop peptide epitope specific to TRACP5a. An immunoassay was devised to identify the so-called “denatured” TRACP using Mab9C5 to capture TRACPs and Mab220-HRP was used to detect 5a form. This assay revealed that all mutant proteins were detectable intracellularly to a similar level (, right, y axis = cells), and that K52T and T89I TRACP were uniquely secreted into the culture medium (, left, y axis = medium) as inactive “denatured” TRACP proteins (). Since WT TRACP is also reactive with Mab9C5, this assay also detects normal immature precursor protein. Our results confirm that all eight TRACP missense mutations result in inactive proteins based on immunochemical assays. It suggests that the destabilization of tertiary structures and misfolding do not allow synthesis of an active enzyme with appropriate protein integrity. Only T89I and M264K were synthesized with intact native epitopes recognized by Mab14G6 and Mab162 and secreted into medium. However, M264K was not detected in the media by immunoassay using Mab9C5. Conversely, K52T was secreted to medium, perhaps as an immature precursor reactive with Mab9C5, but was not reactive to either Mab14G6 or Mab162.

Fig 4

Intracellular expression of recombinant mutant TRACP.

Figs (a) and (b) represent the intracellular 5a, 5b enzyme activity and Figures c and d represent total 5a and 5b protein levels. Figures are depicted with DC, WT as controls along with different mutant cell activities (mutation amino acids are indicated below the X-axis). Details are similar to those indicated in the legend to Fig 3.

Fig 3

Analysis of recombinant mutant TRACP proteins secreted to medium.

Figs (a), (b), (c), and (d) represent the secreted 5a, 5b protein and total protein levels observed (Different mutations tested are indicated on the X-axis). Multiple-group comparisons were performed using one-way analysis of variance (ANOVA) for all samples. The data is presented as mean ± S.E.M. ****, P <0.00001; ***, P <0.0001; Experiments were repeated three times. The Y axis represents the activity observed with different mutant cells in various conditions along with Dendritic cells (DC) and WT as controls.

“Denatured” or precursor mutant TRACP protein expressed by HEK-293 cells.

Denatured TRACP was measured by immunoassay using Mab 9C5 to capture TRACP and Mab 220-HRP was used as secondary antibody to detect bound proteins. Y axis = Medium = Medium supernatants (Left figure); Cells = cell lysates; right figure (protein in samples were normalized to WT type samples; in three separate experiments). Figures represent total protein levels observed with different mutations from medium and cells (mutation amino acids are indicated on X-axis). Details are similar to those indicated in the legend to Fig 3.

Mutant TRACP protein processing

Studies on post translational processing of TRACP mutations carried out by Western blot analysis indicated that WT TRACP exists intracellularly as a mixture of intact 5a-like protein and cleaved 5b protein (, Lanes 2–3) whereas all mutant TRACPs existed solely as uncleaved proteins both in media and cell lysates (, Lanes 4–19). In the top panel () the blot was probed with Mab220 and in the lower blot was probed with Mab9C5. Western blot analysis revealed that the level of secreted TRACP in media was dramatically reduced in all mutations except T89I (; Lane 6 both top and bottom figures). Analysis of mutant K52T by immunoassay and Western blot analysis of secreted TRACP with Mab9C5 did not show any correlation: i.e., it showed a strong signal in immunoassay () and a weak signal in blots (; Lane 4, sample marked as “m” underneath the figure).

Western blot analysis of mutant TRACP proteins secreted to culture media (m) and expressed intracellularly (c).

All mutants express intact 35 kD 5a-like proteins intracellularly; only WT TRACP is present as a processed protein (16 kD fragment shown, Lane 3- WT,c). All mutants are secreted at low levels except T89I, which is secreted in similar amounts to WT. Lanes 8 and 16 in the upper panel, mutants G109R and N262 show mild secretary forms as recognized by Mab220. Duplicate blots were probed with Mab220 (isoform 5a-specific antibody, upper panel) or with Mab 9C5 (5b specific antibody, bottom panel) to detect both intact (5a) and cleaved (5b) TRACP isoforms. Lane 1 indicates the molecular weights of TRACP 5a and 5b. Lanes 2 to 19 indicate samples derived from medium (m) and cells (c).

Deglycosylation of TRACP proteins with Endo H and PNGase F enzymes

We next investigated whether the amino acid substitutions in TRACP mutations, which are not part of the N-glycan acceptor sites (21–25), affected the glycosylation of mutant human TRACPs. The WT and mutant proteins that are secreted into medium have similar glycosylation patterns, similar to the natural TRACP secreted by dendritic cells (DC) (, Lanes 2–4 for DC cells; Lanes 5–7 for WT cells) with both Endo-H sensitive and resistant glycans (; upper figure for TRACP from medium; lower figure for TRACP proteins derived from cells. The recombinant TRACP remaining intracellularly bears more Endo H sensitive glycans compared to natural TRACP, particularly those with mutations (; Lanes 8–31). Mutant K52T has a higher molecular weight compared to all other TRACP proteins (; Lane 8) prior to deglycosylation. However, after removal of oligosaccharides, the polypeptide was the same size as other TRACP proteins (, Lanes 9–10).

Western blot analysis of natural dendritic cell (DC), recombinant WT and mutant samples.

Both DC and WT were used as controls. Mutant TRACP proteins, undigested (C) or after deglycosylation with Endo H enzyme (H, high-mannose and hybrid oligosaccharides) or PNGase F enzyme (F, all N-linked oligosaccharides) were run along with other mutant samples. In the upper panel all mutants faintly demonstrate some bands excepting T89I which showed strong oligomeric bands after deglycosylation. Blots were probed only with Mab9C5 to detect both intact and cleaved forms. Lane 1 indicates the molecular weights of TRACP 5a and 5b (35 kD and 16 kD respectively). Lanes 2 to 31 indicate various samples treated with Endo H and PNGase F (Endo H = H; PNGase F = F).

Limited proteolysis of TRACP proteins by trypsin

In order to study the conversion of proteolytically cleaved inactive mutant TRACP proteins into active TRACP 5b like enzymes, we used trypsin to fragment them along with wild (WT) type TRACP (; Lanes 2–5). Trypsin successfully digested all intracellular mutant TRACP monomeric proteins (; Lanes 6–21). The 16 kD fragment of isoform 5b was clearly evident in trypsin-treated WT samples—Lanes 2 and 3 for TRACP from medium and lanes 4 and 5 for TRACP from cells (intracellular). Although 16 kD TRACP is vaguely visible in T89I, G109R, L201P and M264K mutants (; Lanes 8–13 and 20–21), additional larger proteolytic fragments were also vaguely visible in the M264K mutant (Lanes 21). As expected, proteolysis did not convert TRACP5a to strong TRACP5b bands in blots. There was also no increase in the enzyme activity or pH optima of the TRACP mutants as observed in both at pH 5.2 and 6.1 except for controls samples, DC and WT. Both top and bottom figures () on the left denote untreated controls whereas those on the right () represent trypsin treated samples. These results suggest that the missense mutant proteins (which are probably misfolding) may be more susceptible to trypsin degradation in vitro. Complete degradation of TRACP proteins are clearly visible in lanes 7, 9 11, 13, 15, 17, and 19 even with limited proteolysis with trypsin.

Fig 9

Effect of trypsin treatments on mutant TRACP activities.

Enzyme samples from WT and DC media were used before and after trypsin treatments to estimate TRACP activities in two different pHs as described in Refs. 25 and 26. Controlled trypsin digestion increased the pH optimum from pH 5.2 to 6.1 and increased enzyme activities only in DC and WT samples. Low activities of mutant TRACPs were not affected by trypsin digestions. Experiments were repeated three times. Figs (pH 5.2), (pH 6.1) are untreated and served as controls. Whereas Figs (pH 5.1), (pH 6.1) represent enzyme activities observed after trypsin treatments. Details are similar to those indicated in the legend to Fig 3.

Western blot analysis of WT and mutant TRACP proteins in cell lysates after trypsin treatments.

Undigested control (c) and digested/treated (t) samples were exposed with trypsin (Lanes 2–21). Normal WT samples only show 16 kD forms from both medium (Lanes 2, 3) and cells (Lanes 4, 5) as recognized by Mab9C5. Lane 1 illustrates the molecular weights of TRACP 5a and 5b (35kD and 16kD). Lanes 6 to 21 indicate various mutant samples treated with trypsin. These lanes demonstrate that most of these mutants are incapable of generating 16 kD isoforms unlike WT (Lanes 2–5) samples. Only M264K shows faint multiples bands after fragmentation by trypsin as recognized by Mab9C5 (Lane 21).

Discussion

Tartrate-resistant acid phosphatase plays an important pathophysiologic role in osteoimmunology as evidenced by mice bearing loss-of-function mutations in ACP5 [20, 21]). As the single known cause for SPENCD, a specific human disease with a bone and immune phenotype, loss-of-function TRACP mutations represent a proof-of-principle experiment of nature [12, 13]. Patients with homozygous or compound heterozygous mutations display a variable degree of skeletal dysplasia, variable levels of neurological deficit, one or more autoimmune phenotypes and bone tumors [7, 8, 12, 13, 39]. Recently, it was discovered that TRACP may also have pathological consequences when over-expressed [24]. Functional genomics have implicated ACP5 as one among six genes with verified oncogenic and pro-invasive capability in human malignant melanoma [24]. These clinical consequences of ACP5 as a result of under and over expression imply that ACP5 is tightly regulated. Computer modelling predicts that the ACP5 missense mutations in SPENCD could lead to a destabilized protein structure, but direct analysis of mutant TRACP proteins have not been done so far. Hence, the results of these studies with eight mutant TRACP proteins are reported in this paper. The present study examines the effects of single amino acid substitutions related to SPENCD on protein biosynthesis in vivo and its enzyme activity (both intracellular and secreted isoforms). It also sheds light on the specificity of the anti-TRACP monoclonal antibodies used in these studies and identifies some potential structural targets for inactivation of TRACP in instances where ACP5 is pathologically over-expressed during oncogenesis.

TRACP5b remains in the intracellular endosomal compartment in most natural sources and in recombinant TRACP expressing cells, except osteoclasts [25, 26]. Osteoclasts uniquely release TRACP5b with other bone matrix products by transcytosis of endosomes during bone resorption [6]. The individual amino acid substitutions in mutant TRACP associated with SPENCD exhibited similar effects on activity and gross structure, but also had some variable effects on protein conformation as revealed by the pattern of immuno-reactivity with different antibodies (this study). First, all missense mutations caused inhibition of enzymatic activity either due to misfolding or modified structures. Second, immunoassays utilizing antibodies reactive to native conformational determinants showed that most mutants are undetectable in both cells and medium; with T89I and M264K being the exceptions. However, immuno-histochemical staining, Western blot analysis and immunoassays utilizing antibodies reactive with linear, denatured determinants/epitopes revealed that all mutant TRACPs were expressed abundantly intracellularly but were not processed by HEK-293 cells into highly active, heterodimeric secretory isoform 5b-like TRACP.

Immunoassays for native TRACP5a revealed that only T89I and M264K are secreted into medium as inactive proteins, whereas immunoassays for denatured TRACP5a showed abundant secretion of K52T and T89I, but not M264K. Immunoblots of secreted TRACP with Mab220 and Mab9C5 both showed strong signals with T89I only. All other mutants were present in media in trace amounts. The anomalies causing method-based differential detection of the secreted forms of K52T and M264K remain unexplained. Since there is ample production of some of the mutants without enzyme activity, misfolding of proteins must therefore account for their inability to exert enzymatic functions. In all, five out of eight ACP5 mutations associated with SPENCD disease drastically interfere with TRACP exiting the cell. This explains the inability to detect TRACP protein in the serum of SPENCD patients [12, 13], or TRACP activity in their dendritic cells [13].

The site and type of glycosylation on TRACP is variable depending on the species and source of enzyme [34-36], and they have been assigned different functional roles. Human serum TRACP5a was originally defined by the presence of sialic acid [35]. Sialylation generally provides stability for a glycoprotein enzyme (TRACP) destined for secretion and extracellular function(s) [37]. Using a recombinant rat TRACP expression system, Wang et al. [35] demonstrated that at least one oligosaccharide is necessary for TRACP protein stability. In the present study all intracellular mutant TRACPs were N-glycosylated primarily with Endo H sensitive glycans (, Lanes 8–31) whereas WT and DC TRACPs bore Endo-H resistant glycans (, Lanes 2–4 especially for DC cells. Secreted TRACPs from all sources were similarly glycosylated in intact isoform-5a like proteins. The reduced amount of oligosaccharide processing and secretion of mutant TRACPs would be consistent with their destabilized structures. Failure to efficiently route to the Golgi for processing to add complex oligosaccharides will terminate eventual secretion.

Proteolytic processing of the loop peptide of monomeric TRACP is a critical mechanism for regulating enzymatic activity in vivo and in vitro [4, 5]. Limited proteolytic digestion with trypsin () was ineffective in converting the inactive mutant TRACPs to active enzymes. Proteolysis of the mutant proteins () may be responsible to generate inactive TRACP forms as measured for enzyme activity both at their optimal pH, 5.2 and 6.1. In all, the ACP5 missense mutations associated with SPENCD generally lead to improper conformation, inefficient glycosylation, interrupted intracellular trafficking, and limited secretion. This finding explains the lack of detectable TRACP activity and protein in SPENCD patient sera and cells. Few mutant TRACP proteins that are secreted in vitro, for example T89I, probably do not survive mechanisms for removal of abnormal glycoproteins in vivo. It is uncertain why these mutant proteins with presumed abnormal structure escape intracellular degradation. Perhaps, we are detecting only a fraction of protein in transit during a continuous flux of precursor mutant TRACPs from the endoplasmic reticulum to the proteasome, without normal post-translational processing and endosomal compartmentalization of active isoform 5b or secretion of less active isoform 5a. Our findings contribute directly to understanding more completely the structure-function relationships in TRACP biochemistry. Further work on structural analysis of each TRACP mutant will reveal its precise functional relevance.

TRACP is emerging as a key pathophysiological component in human diseases, both when decreased or increased [24, 38]. Control of TRACP levels, its activity and compartmentalization are required for homeostasis in skeletal metabolism and immune responsiveness, and perhaps oncogenesis [24, 39, 40]. Therefore, research into the role of TRACP in immune regulatory circuits and the direct pathological effects of TRACP deficiency or over-expression should be rewarding. More detailed molecular analysis of mutant TRACP proteins could lead to design of specific drugs targeting TRACP. Currently in the pipe-line are drugs such as PSTP-3, 5-Me and Tiliroside, which target TRACP for osteoporosis [41, 42]. Also, appreciation of the direct effects of ACP5 gene expression on diseases and pathways involving TRACP could lead to compounds that augment or mitigate these pathways upstream of TRACP [43].

(PDF)

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18 Aug 2019

PONE-D-19-20746

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

PLOS ONE

Dear Dr. Venugopal,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Dr. Sakamuri V. Reddy

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Major Comments:

1) In Results Section the Figures are not cited sequentially. It is cited like Fig. 2, 3 then 6A, 6B, Fig 3, then Fig. 7, Fig.4, Fig 5 and Fig. 8. Authors need to completely revise the Results section and explain the results according to the sequence of the figures or change the Figure numbers.

2) Figure legends are not written properly and completely. For example, in many Figure legends the results are explained and then experimental detail. There is no need to write the results in Figure legends. Please write the experimental details and number of repeat experiments.

3) Figures 6a and 6b should be numbered as Fig. 6 and Fig. 7 respectively.

3) It is not clear that why all mutants TRACPs were detected intracellularly to a similar level? Please discuss.

3) Discussion is lengthy. It should be shortened.

Minor comments:

There are some typo and grammatical errors, that need to be corrected.

Reviewer #2: PONE-D-19-20746

Characterization of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatases Associated with Spondyloenchondrodysplasia

This paper characterizes the Acp5 mutations encoding TRACP that commonly associated with Spondyloenchondrodysplasia (SPENCD). SPENCD is a recently identified disease, and it seems promising and exciting to investigate such disorder at the molecular level.

However, while reading the MS, I felt lost. Data is impressive. Clearer presentation is needed. The overall idea was not well-explained. MS needs rewriting. Some of the general and specific comments are stated below:

• General comments:

o The overall purpose of this study was not stated clearly.

o Excessive explanation in the result section. This makes some sorts of confusion. Every result subsection should plainly and simply state the result, whereas any other explanations should be carried out to the "discussion” section.

o There should be more introductions about Spondyloenchondrodysplasia and TRACP proteins in general. The relationship between the diseases and TRACP has to be elaborated in much detail.

o This reviewer is not clear about the bands in the Western analyses. MW markers are not provided. Several lanes are provided with no lane numbers.

o How do you normalize the secreted and intracellular proteins? Why do you think the top band as a non-specific band and consider that as a loading control? For secreted proteins in media, it is essential to show a Coomassie blue stained gel as a loading control. GAPDH or any other intracellular loading control should be added for normalization of the intracellular proteins

• Specific comments:

o The underlying reason for using HEK-293 cell line is not stated at all. Why not using other cell lines that not ubiquitously expressing TRACP?

o Figure 2: No clear illustration or indication of the stainable activity of cells. Quantification would also help.

o Result: the result section should correspond with the numerical order of the figures. The readers must not jump between figures while reading.

o Results: figure 6B does not correlate with what mentioned in the corresponding result section.

o Figures: Western analyses: comments provided under general comments

o Figures: Quantification alone for figure 7 is not sufficient. It is better to provide visual representations such as immunostaining and Western blotting analyses.

**********

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Reviewer #1: No

Reviewer #2: No

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5 Nov 2019

The authors profusely thank the reviewers’ for their constructive and valuable criticism which we strongly believe making our draft paper more refined and may be acceptable at this time. We earnestly apologize for typos, errors and delay in responding to review as our labs are being shifted from one campus to the other which made “our hands tied” to respond to all the recommendations. As the authors emanate from a non-English speaking country, we still learning and refining our way of writing. Hope we will get better in the future and certainly referees’ recommendations are tremendous helpful to refine our paper. This time we have given our manuscript to an English professor who was kind enough to correct the grammatical errors and typos. Figures which are ambiguous and “not scientifically talking back” are either removed or renumbered.

Again the authors thank the referees and we feel at this time, this revised manuscript should be acceptable in PLOSOne, which we consider as highest honor to publish from the east end. We will be happy to modify further if needed. Details on SPENCD disorder related abnormalities are coming up sharply in the last decade.

Referee 1:

Authors profusely thank the Referee 1 for the constructive and immensely helpful criticisms.

1. We surely apologize for the conspicuous errors in the Results section for placing the figure numbers upside down. As recommended we now either correctly and sequentially place or change the figure numbers. Entire Result section was rewritten eliminating unwanted details so that both figures and results will coincide with each other.

2. As rightly pointed out by the Referee, figure legends are rewritten so that they are devoid of experimental details. Experimental repeats were also mentioned.,

3. As recommended the figures 6a and 6b were renumbered as 6 and 7.

4. With regard to the observation of similar levels of intracellular TRACP mutant proteins.

We have discussed in appropriate section with regard to the similar levels of TRACP mutant proteins (Results and Discussion as well). First, we did all the experiments with stably transfected cell lines which took a large amount of time to perfect in between experiments. Realizing the cell number variations in different experiments may generate spurious values, we culture exact same number of cells per flask by counting both before and after culturing. This technique largely eliminated the quantitative variations between experiments, and we were able to do this as our personnel are fully trained and routinely do these techniques for hematology labs.

5. As recommended the discussion was sharply shortened and now it should be suffice.

6. Most of our typos and grammatical errors were corrected with the help of an English professor. As the authors are from a non-English speaking country we still evolving and hope umbrage will not be taken for this inability and deficiency.

Referee 2:

1. Overall aim of the paper is now mentioned in the introduction.

2. As recommended excessive explanation in the Results section was eliminated in toto and discussion section was also modified and shortened.

3. Relationship between SPENCD and TRACP was also elaborated in the Introduction.

4. Molecular weight of TRACP isoforms 5a and 5b in the Western blots were indicated now. Putting together to make a meaningful picture was a herculean task for us and therefore we have used professional help to put together our multiple pictures (using Adobe Photoshop by Ms. Barbara J, please vide acknowledgement). We normally run standard proteins markers and to put together multiple runs at different points with standard marker proteins (which have small difference in migration and therefore they were removed). Now we marked the molecular weights of 5a and 5b proteins as they are the only primary bands of interest.

Thanking you.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Dec 2019

PONE-D-19-20746R1

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

PLOS ONE

Dear Dr. Venugopal,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR: I suggest the authors to proof read the manuscript for English language and follow the journal format as noted in the reviewers comments below. Please address the  several minor comments raised by rev#3 carefully.

We would appreciate receiving your revised manuscript by Jan 18 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Dr. Sakamuri V. Reddy

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: There is only slight improvement in the revised manuscript.

Major issues:

1) Authors have not provided point-by-point response in detail. Very casual approach is taken by authors in writing of the response to reviewers comments.

2) There is only slight improvement in English writing in the manuscript.

3) However, the scientific language is still very poor throughout the manuscript, particularly the authors have not used proper scientific terms and framing of the sentences and grammar. It appears that manuscript is not properly edited by senior authors.

3) Result section is still very poor. Results of all the experiments are not written in details. The sub-figures are not cited in the text. Although, there are several sub-figures in many figures such as Figures 3, 4, 5 and 9, authors have just written there numbers.

4) Many Figures Legends are incomplete. e.g. In Figure 1 Legend, it is mentioned that missence mutations are shown below the WT sequence. This is not reflected in actual figure. Similarly, there are many mistakes in other Figures legends.

Reviewer #2: PONE-D-19-20746R1

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid

Phosphatase Associated with Spondyloenchondrodysplasia

The MS has been revised as suggested, and the authors corrected the crucial issues.

It can be accepted for publication

Reviewer #3: Comments to the Author:

This article attempt to characterize the eight already reported missense mutations in Acp5 which is associated with spondyloenchondrodysplasia (SPENCD). The authors identified that the presence of mutants TRACP proteins are unable to induce enzymatic activity. Further, except T89I and M264K mutant proteins, others are in denatured precursor forms. The authors concluded that understanding the structural and functional aspects of mutant TRACP could lead to better understating of immune response and bone metabolism as well as targeting drug development.

The concept of the paper is relevant however there are many concerns that should be addressed before publication. The authors should follow the scientific writing format rather than common words and sentences. The whole manuscript should be corrected for details, clarity, and grammar.

Major issues:

1) In Abstract: RT-qPCR analysis is mentioned but there is no such figure and result in appropriate sections.

2) Methods section written poorly, refer the articles and follow the formats. For instance, western blotting is written without clarity and details. Some of the Methods written like results that should be avoided.

3) It is not appropriate to write a sentence like this in the result section “Other salient features of TRACP are described in Figure 1 legend”. Provide more details about TRACP enzyme in this section.

4) “The steady state transcription ….10-fold lower compared to WT”. There is no corresponding figures/results in this manuscript.

Minor issues:

1) In Abstract: At the end, summarize or conclude the work and it should exactly reflect the results. Then write what it could be useful to, such as drug development and underlying mechanisms, etc.

2) In Abstract: “Certainly, determining the structure functional relationships in TRACP will expand….” Should modify to “Determining the structural and functional relationships of TRACP may expand… “

3) An unscientific sentence like this and many others throughout the manuscript should be modified “TRACP has many functions in vitro and its biological role in bone resorption and immune responses is becoming clearer”. What exactly authors want to convey with this sentence “TRACP has many functions in vitro….”

4) “Therefore, serum TRACP5a may serve as a marker for systemic macrophage number…” is it possible to count the macrophage number using serum TRACP5a level? Similarly, isoform 5b release and osteoclast number? Clarify both the statements.

5) Mentioned in this sentence that “We and others have shown earlier [9, 10] that mutations…” but there is one other’s paper cited, add more reference or modify.

6) Follow the same format for gene name, ACP5 or Acp5.

7) Add references to these sentences and many others "More specifically, ….TRACP enzyme”, “Other salient features…..calcification”

8) “Transfection of synthetic genes” This subtitle should be written like this “Transformation of mutant genes and subcloning” and describe the methods in the text.

9) “Transient transfection and generation of stable cell line” This should be a subtitle for the next paragraph (from this sentence onwards “HEK-293 cells were transfected with……recommended procedures.”

10) What is the loading control used for western blot analysis? Explain.

11) Figures should be labeled appropriately. For instance, Fig 2, TRAP staining and Mab220.

12) Y-axis scale difference is high between min and max in many bar graphs, Therefore if it appropriate to represent the graph with the broken y-axis to show the lower values.

13) The results section should be written clearly with details.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

13 Feb 2020

PONE-D-19-20746R1

The authors profusely thank the reviewers for their constructive and valuable criticism which we strongly believe has enhanced the quality of the revised paper. We apologize for all errors in the earlier version. As recommended, we have now revised our manuscript according to the reviewers’ suggestions and we have tried our best by giving detailed responses to each of the reviewers’ questions point-by-point.

Reviewer #1: There is only slight improvement in the revised manuscript.

Major issues:

1) Authors have not provided point-by-point response in detail. Very casual approach is taken by authors in writing of the response to reviewers’ comments.

Reply to the Reviewer 1: We apologize for the conspicuous errors in the Results section. We have added more information and rewritten the results section in better English.

2) There is only slight improvement in English writing in the manuscript.

Reply to the Reviewer 1: As recommended, we have now corrected the entire manuscript especially with regard to English and grammar.

3) However, the scientific language is still very poor throughout the manuscript, particularly the authors have not used proper scientific terms and framing of the sentences and grammar. It appears that manuscript is not properly edited by senior authors.

Reply to the Reviewer 1: As recommended several changes have been made throughout the manuscript with proper scientific terms and framing the sentences in better English.

3) Result section is still very poor. Results of all the experiments are not written in details. The sub-figures are not cited in the text. Although, there are several sub-figures in many figures such as Figures 3, 4, 5 and 9, authors have just written there numbers.

Reply to the Reviewer 1: Results section has been drastically modified and details added wherever necessary. As recommended, Discussion was also drastically shortened. The sub-figures have been cited in the text. We have revised the figure numbers and attached as individual files as recommended.

4) Many Figures Legends are incomplete. e.g. In Figure 1 Legend, it is mentioned that missense mutations are shown below the WT sequences. This is not reflected in actual figure. Similarly, there are many mistakes in other Figures legends.

Reply to the Reviewer 1: We have revised and rewritten the figure legends as recommended.

Figure 1 was carefully revised to reflect the details of TRACP.

Reviewer #2:

Characterization of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid

Phosphatase Associated with Spondyloenchondrodysplasia The MS has been revised as suggested, and the authors corrected the crucial issues. It can be accepted for publication

Reply to the Reviewer 2: We thank you for your suggestions.

Reviewer #3:

This article attempts to characterize the eight already reported missense mutations in Acp5 which is associated with spondyloenchondrodysplasia (SPENCD). The authors identified that the presence of mutants TRACP proteins are unable to induce enzymatic activity. Further, except T89I and M264K mutant proteins, others are in denatured precursor forms. The authors concluded that understanding the structural and functional aspects of mutant TRACP could lead to better understating of immune response and bone metabolism as well as targeting drug development. The concept of the paper is relevant however there are many concerns that should be addressed before publication. The authors should follow the scientific writing format rather than common words and sentences. The whole manuscript should be corrected for details, clarity, and grammar.

Major issues:

1) In Abstract: RT-qPCR analysis is mentioned but there is no such figure and

result in appropriate sections.

Reply to the Reviewer 3: Since the contribution by RT-qPCR study is minimal we have removed this section from our paper and changed all sections accordingly.

2) Methods section written poorly, refer the articles and follow the formats. For

instance, Western blotting is written without clarity and details. Some of the Methods

written like results that should be avoided.

Reply to the Reviewer 3: As recommended we have revised our Methods section and added more details to Western blot technique. Results reported in Methods section have been removed as per suggestion.

3) It is not appropriate to write a sentence like this in the result section “Other salient features of TRACP are described in Figure 1 legend”. Provide more details about TRACP enzyme in this section.

Reply to the Reviewer 3: As suggested, we have provided details about TRACP enzyme in this Results section with Figure 1.

4) “The steady state transcription…..10 fold lower compared to WT”. Thereis no corresponding figures/results in this manuscript.

Reply to the Reviewer 3: As RT-qPCR has been removed in this revised version, sentences related to this work have also been removed from other sections of this resubmission.

Minor issues:

1) In Abstract: At the end, summarize or conclude the work and it should exactly reflect the results. Then write what it could be useful to, such as drug development and underlying mechanisms, etc.

Reply to the Reviewer 3: As recommended, we have completely rewritten the Abstract..

2) In Abstract: “Certainly, determining the structure functional relationships in TRACP will expand….” Should modify to “Determining the structural and functional relationships of TRACP may expand… “

Reply to the Reviewer 3: As recommended, we have modified these sentences as “Determining the structural and functional relationships of TRACP may expand…”.

3) An unscientific sentence like this and many others throughout the manuscript should be modified “TRACP has many functions in vitro and its biological role in bone resorption and immune responses is becoming clearer”. What exactly authors

want to convey with this sentence “TRACP has many functions in vitro….”

Reply to the Reviewer 3: As recommended, we have modified this sentence. Please see our revised manuscript.

4) “Therefore, serum TRACP5 may serve as a marker for systemic macrophage number….” Is it possible to count the macrophage number using serum TRACP5a level? Similarly, isoform 5b release and osteoclast number?? Clarify both statements.

Reply o the Reviewer 3: We have modified the sentence with clarity in this revised version.

5) Mentioned in this sentence that “We and others have shown earlier [9, 10] that mutations…” but there is one other’s paper cited, add more reference or modify.

Reply to the Reviewer 3: As recommended, we have now added more references here.

6) Follow the same format for gene name, ACP5 or Acp5.

Reply to the Reviewer 3: We apologize for this inconsistency. We have consistently followed the same pattern of human gene nomenclature. Human gene should be in caps and we have changed accordingly throughout the manuscript.

7) Add references to these sentences and many others "More specifically, ….TRACP enzyme”, “Other salient features…. calcification”

Reply to the Reviewer 3: As suggested, we have now added references to these sentences.

8) “Transfection of synthetic genes” This subtitle should be written like this “Transformation of mutant genes and subcloning” and describe the methods in the text.

Reply to the Reviewer 3: As directed, the subtitle has been changed accordingly.

9) “Transient transfection and generation of stable cell line” This should be a subtitle for the next paragraph (from this sentence onwards “HEK-293 cells were transfected with……recommended procedures.”

Reply to the Reviewer 3: As suggested, the subtitle has been inserted in its place.

10) What is the loading control used for Western blot analysis? Explain

Reply to the Reviewer 3: Since there are no other proteins present in the blot other than our electrophoretically homogeneous TRACP proteins, we treat this protein as loading control. Also the electrophoresis combs are not sufficient enough to increase the number of samples.

11) Figures should be labeled appropriately. For instance, Fig 2, TRAP staining and Mab220.

Reply to the Reviewer 3: We thank the reviewer for the comments. As rightly pointed out by the Referee, figures have been rewritten appropriately, and the figure legends have also been rewritten so that they are devoid of experimental details.

12) Y-axis scale difference is high between min and max in many bar graphs, therefore if it appropriate to represent the graph with the broken y-axis to show the lower values.

Reply to the Reviewer 3: We have revised the graphs as much as the Software package allows.

13) The results section should be written clearly with details.

Reply to the Reviewer 3: We have corrected the Results section with clarity now.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Feb 2020

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

PONE-D-19-20746R2

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Reviewer #3: All comments have been addressed

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Reviewer #1: Sub figures such as A, B, C etc.of Figures 4, 5 are not yet cited in Resullts. That need to be corrected.

Reviewer #3: (No Response)

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9 Mar 2020

PONE-D-19-20746R2

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

Dear Dr. Venugopal:

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