Biochemical and sequence similarities between the TIGR/myocilin and olfactomedin protein families and the effects of TIGR in primary open angle glaucoma

Glaucoma is the leading cause of blindness in African-Americans and the second leading cause of blindness worldwide. Early detection of the disease could significantly reduce these devastating effects. Following the isolation of the TIGR/MYOC protein in a steroid-induced glaucoma model, mutations in the gene were found to explain a number of cases of open-angle glaucoma. The structure and function of the TIGR protein are unknown; hence, computational sequence analysis techniques were used to identify important regions of the protein and to guide experimental efforts.; Starting with the proposed relationship between TIGR's C-terminal domain and the olfactomedin family, analysis of the individual protein domains and conserved motifs within the families demonstrated additional homology between TIGR and olfactomedin in the N-terminal domain. The exon 2 region of the TIGR molecule was found to be similar to a bacteria] metalloprotease inhibitor, and not to the olfactomedin family, suggesting this region may result from an insertion event.; Next, “sequence walk” techniques and the Shotgun congruence analysis program, were used to identify a set of 31 olfactomedin-domain-containing (ODC) sequences, including the closely related TIGR and olfactomedin orthologs. A subset of the 31 ODC sequences includes related domains N-terminal to the olfactomedin domain. These domains include several highly conserved cysteines involved in intermolecular disulfide bonds and a residual leucine zipper, as well as, highly similar predicted secondary structures. Additional conserved motifs were identified in the C-terminal domain of the ODC proteins. By comparing these conservations to known mutations and polymorphisms in human TIGR, two regions that may be key to the successful structure/function of the TIGR molecule were identified. A potential distantly related superfamily member (chitinase A) was also identified based on the conserved ODC motifs.; To investigate the contribution of cysteines within each exon region to the aggregative properties of TIGR, peptides were expressed including one or two of the individual exon regions of the protein. Western blots of the peptides treated under reducing and non-reducing conditions confirmed the importance of N- and C-terminal cysteines in intermolecular disulfide bonds and provided further evidence supporting our proposed model of TIGR oligomerization.