Birgit Eisenhaber1,2, Swati Sinha3, Chaitanya K Jadalanki3, Vladimir A Shitov3,4, Qiao Wen Tan3,5, Fernanda L Sirota3, Frank Eisenhaber6,7,8. 1. Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore, 138671, Republic of Singapore. birgite@bii.a-star.edu.sg. 2. Genome Institute of Singapore (BII), Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore. birgite@bii.a-star.edu.sg. 3. Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore, 138671, Republic of Singapore. 4. Siberian State Medical University, Moskovskiy Trakt, 2, Tomsk, Tomsk Oblast, 634050, Russia. 5. School of Biological Science (SBS), Nanyang Technological University (NTU), 60 Nanyang Drive, Singapore, 637551, Republic of Singapore. 6. Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore, 138671, Republic of Singapore. franke@bii.a-star.edu.sg. 7. Genome Institute of Singapore (BII), Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore. franke@bii.a-star.edu.sg. 8. School of Biological Science (SBS), Nanyang Technological University (NTU), 60 Nanyang Drive, Singapore, 637551, Republic of Singapore. franke@bii.a-star.edu.sg.
Abstract
BACKGROUND: The human proteins TMTC1, TMTC2, TMTC3 and TMTC4 have been experimentally shown to be components of a new O-mannosylation pathway. Their own mannosyl-transferase activity has been suspected but their actual enzymatic potential has not been demonstrated yet. So far, sequence analysis of TMTCs has been compromised by evolutionary sequence divergence within their membrane-embedded N-terminal region, sequence inaccuracies in the protein databases and the difficulty to interpret the large functional variety of known homologous proteins (mostly sugar transferases and some with known 3D structure). RESULTS: Evolutionary conserved molecular function among TMTCs is only possible with conserved membrane topology within their membrane-embedded N-terminal regions leading to the placement of homologous long intermittent loops at the same membrane side. Using this criterion, we demonstrate that all TMTCs have 11 transmembrane regions. The sequence segment homologous to Pfam model DUF1736 is actually just a loop between TM7 and TM8 that is located in the ER lumen and that contains a small hydrophobic, but not membrane-embedded helix. Not only do the membrane-embedded N-terminal regions of TMTCs share a common fold and 3D structural similarity with subgroups of GT-C sugar transferases. The conservation of residues critical for catalysis, for binding of a divalent metal ion and of the phosphate group of a lipid-linked sugar moiety throughout enzymatically and structurally well-studied GT-Cs and sequences of TMTCs indicates that TMTCs are actually sugar-transferring enzymes. We present credible 3D structural models of all four TMTCs (derived from their closest known homologues 5ezm/5f15) and find observed conserved sequence motifs rationalized as binding sites for a metal ion and for a dolichyl-phosphate-mannose moiety. CONCLUSIONS: With the results from both careful sequence analysis and structural modelling, we can conclusively say that the TMTCs are enzymatically active sugar transferases belonging to the GT-C/PMT superfamily. The DUF1736 segment, the loop between TM7 and TM8, is critical for catalysis and lipid-linked sugar moiety binding. Together with the available indirect experimental data, we conclude that the TMTCs are not only part of an O-mannosylation pathway in the endoplasmic reticulum of upper eukaryotes but, actually, they are the sought mannosyl-transferases.
BACKGROUND: The human proteins TMTC1, TMTC2, TMTC3 and TMTC4 have been experimentally shown to be components of a new O-mannosylation pathway. Their own mannosyl-transferase activity has been suspected but their actual enzymatic potential has not been demonstrated yet. So far, sequence analysis of TMTCs has been compromised by evolutionary sequence divergence within their membrane-embedded N-terminal region, sequence inaccuracies in the protein databases and the difficulty to interpret the large functional variety of known homologous proteins (mostly sugar transferases and some with known 3D structure). RESULTS: Evolutionary conserved molecular function among TMTCs is only possible with conserved membrane topology within their membrane-embedded N-terminal regions leading to the placement of homologous long intermittent loops at the same membrane side. Using this criterion, we demonstrate that all TMTCs have 11 transmembrane regions. The sequence segment homologous to Pfam model DUF1736 is actually just a loop between TM7 and TM8 that is located in the ER lumen and that contains a small hydrophobic, but not membrane-embedded helix. Not only do the membrane-embedded N-terminal regions of TMTCs share a common fold and 3D structural similarity with subgroups of GT-C sugar transferases. The conservation of residues critical for catalysis, for binding of a divalent metal ion and of the phosphate group of a lipid-linked sugar moiety throughout enzymatically and structurally well-studied GT-Cs and sequences of TMTCs indicates that TMTCs are actually sugar-transferring enzymes. We present credible 3D structural models of all four TMTCs (derived from their closest known homologues 5ezm/5f15) and find observed conserved sequence motifsrationalized as binding sites for a metal ion and for a dolichyl-phosphate-mannose moiety. CONCLUSIONS: With the results from both careful sequence analysis and structural modelling, we can conclusively say that the TMTCs are enzymatically active sugar transferases belonging to the GT-C/PMT superfamily. The DUF1736 segment, the loop between TM7 and TM8, is critical for catalysis and lipid-linked sugar moiety binding. Together with the available indirect experimental data, we conclude that the TMTCs are not only part of an O-mannosylation pathway in the endoplasmic reticulum of upper eukaryotes but, actually, they are the sought mannosyl-transferases.
Authors: José Juan Almagro Armenteros; Konstantinos D Tsirigos; Casper Kaae Sønderby; Thomas Nordahl Petersen; Ole Winther; Søren Brunak; Gunnar von Heijne; Henrik Nielsen Journal: Nat Biotechnol Date: 2019-02-18 Impact factor: 54.908
Authors: James Alexander Baker; Wing-Cheong Wong; Birgit Eisenhaber; Jim Warwicker; Frank Eisenhaber Journal: BMC Biol Date: 2017-08-18 Impact factor: 7.431
Authors: Mark Johnson; Irena Zaretskaya; Yan Raytselis; Yuri Merezhuk; Scott McGinnis; Thomas L Madden Journal: Nucleic Acids Res Date: 2008-04-24 Impact factor: 16.971
Authors: Sara El-Gebali; Jaina Mistry; Alex Bateman; Sean R Eddy; Aurélien Luciani; Simon C Potter; Matloob Qureshi; Lorna J Richardson; Gustavo A Salazar; Alfredo Smart; Erik L L Sonnhammer; Layla Hirsh; Lisanna Paladin; Damiano Piovesan; Silvio C E Tosatto; Robert D Finn Journal: Nucleic Acids Res Date: 2019-01-08 Impact factor: 16.971
Authors: Caitríona E McInerney; Joanna A Lynn; Alan R Gilmore; Tom Flannery; Kevin M Prise Journal: Curr Issues Mol Biol Date: 2022-07-02 Impact factor: 2.976