Deletion of cysteine 369 in lysyl hydroxylase 1 eliminates enzyme activity and causes Ehlers-Danlos syndrome type VI.

This study describes the relative contribution of the 10 cysteine residues in lysyl hydroxylase 1 (LH1) to enzyme activity. We have identified a novel mutation of a 15-bp deletion in exon 11 in one LH1 allele, that codes for amino acids 367-371 (DLCRQ), in two unrelated compound heterozygous patients with Ehlers-Danlos type VI. The mutations in their other alleles were a C1119T change (exon 10) and a predicted Q49X (exon 2). We confirmed that the loss of cysteine 369 in the deleted sequence contributed to the diminished enzyme activity by structure/function analysis of mutant LH1 constructs, in which C369 and the nine other cysteines were individually mutated to serine by site-directed mutagenesis of a normal pAcGP67/LH1cDNA construct. Following their expression in an Sf9 insect cell/baculovirus system, SDS-PAGE and Western analysis showed that equivalent levels of correctly-sized (85-kDa) products were secreted. The mutation of residues C369 and also C375, C552 and C687 virtually eliminated LH activity, whereas mutations of C267, C270, and C680 had an intermediate effect. In contrast, the C204S, C484S and C566S constructs had normal activity. Although disulfide bond formation may affect the relative contribution of each cysteine to LH activity, catalytic activity does not appear to be directly related to dimerization of the enzyme.