A Multidomain TIGR/Olfactomedin Protein Family with Conserved Structural Similarity in the N-terminal Region and Conserved Motifs in the C-terminal Region
2002; Elsevier BV; Volume: 1; Issue: 5 Linguagem: Inglês
10.1074/mcp.m200023-mcp200
ISSN1535-9484
AutoresMichelle Green, Teri E. Klein,
Tópico(s)Retinal Development and Disorders
ResumoBased on the similarity between the TIGR (trabecular-meshwork inducible glucocorticoid response) (also known as myocilin) and olfactomedin protein families identified throughout the length of the TIGR protein, we have identified more distantly related proteins to determine the elements essential to the function/structure of the TIGR and olfactomedin proteins. Using a sequence walk method and the Shotgun program, we have identified a family including 31 olfactomedin domain-containing sequences. Multiple sequence alignments and secondary structure analyses were used to identify conserved sequence elements. Pairwise identity in the olfactomedin domain ranges from 8 to 64%, with an average pairwise identity of 24%. The N-terminal regions of the proteins fall into two subgroups, one including the TIGR and olfactomedin families and another group of apparently unrelated domains. The TIGR and olfactomedin sequences display conserved motifs including a residual leucine zipper region and maintain a similar secondary structure throughout the N-terminal region. The correlation between conserved elements and disease-associated mutations and apparent polymorphisms in human TIGR was also examined to evaluate the apparent importance of conserved residues to the function/structure of TIGR. Several residues have been identified as essential to the function and/or structure of the human TIGR protein based on their degree of conservation across the family and their implication in the pathogenesis of primary open-angle glaucoma. Additionally, we have identified a group of chitinase sequences containing several of the highly conserved motifs present in the C-terminal region of the olfactomedin domain-containing sequences. Based on the similarity between the TIGR (trabecular-meshwork inducible glucocorticoid response) (also known as myocilin) and olfactomedin protein families identified throughout the length of the TIGR protein, we have identified more distantly related proteins to determine the elements essential to the function/structure of the TIGR and olfactomedin proteins. Using a sequence walk method and the Shotgun program, we have identified a family including 31 olfactomedin domain-containing sequences. Multiple sequence alignments and secondary structure analyses were used to identify conserved sequence elements. Pairwise identity in the olfactomedin domain ranges from 8 to 64%, with an average pairwise identity of 24%. The N-terminal regions of the proteins fall into two subgroups, one including the TIGR and olfactomedin families and another group of apparently unrelated domains. The TIGR and olfactomedin sequences display conserved motifs including a residual leucine zipper region and maintain a similar secondary structure throughout the N-terminal region. The correlation between conserved elements and disease-associated mutations and apparent polymorphisms in human TIGR was also examined to evaluate the apparent importance of conserved residues to the function/structure of TIGR. Several residues have been identified as essential to the function and/or structure of the human TIGR protein based on their degree of conservation across the family and their implication in the pathogenesis of primary open-angle glaucoma. Additionally, we have identified a group of chitinase sequences containing several of the highly conserved motifs present in the C-terminal region of the olfactomedin domain-containing sequences. Our previous work has established that the relationship between the TIGR (trabecular-meshwork inducible glucocorticoid response) (also known as myocilin) and olfactomedin proteins is evident not only in the C-terminal olfactomedin domain but also in the N-terminal domain of both proteins (1.Green M.L. Do H. Polansky J.R. Nguyen T.D. Klein T.E. Similarities and differences between the TIGR and olfactomedin proteins.Invest. Ophthalmol. Vis. Sci. 2001; 42 (Abstr. 3531): 656Google Scholar). Although the N-terminal domain of olfactomedin is divergent (2.Karavanich C.A. Anholt R.R. Molecular evolution of olfactomedin.Mol. Biol. Evol. 1998; 15: 718-726Google Scholar), the C-terminal domain is highly conserved among the TIGR and olfactomedin proteins. To identify sequence elements important to the structure and function of this family in a broader context, a more divergent set of sequences must be evaluated (3.Babbitt P. Gerlt J. Understanding enzyme superfamilies. Chemistry as the fundamental determinant in the evolution of new catalytic activities.J. Biol. Chem. 1997; 272: 30591-30594Google Scholar). Karavanich and Anholt (2.Karavanich C.A. Anholt R.R. Molecular evolution of olfactomedin.Mol. Biol. Evol. 1998; 15: 718-726Google Scholar) first compared the olfactomedin-related proteins from rat, mouse, human, and frog and identified several conserved motifs. The pairwise identities among these sequences ranged from 22 to 98% identical. Upon identification of the human TIGR protein's homology with olfactomedin, Nguyen et al. (4.Nguyen T.D. Chen P. Huang W.D. Chen H. Johnson D. Polansky J.R. Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells.J. Biol. Chem. 1998; 273: 6341-6350Google Scholar) confirmed that TIGR possessed several of the motifs identified by Karavanich and Anholt (2.Karavanich C.A. Anholt R.R. Molecular evolution of olfactomedin.Mol. Biol. Evol. 1998; 15: 718-726Google Scholar). Rozsa et al. (5.Rozsa F.W. Shimizu S. Lichter P.R. Johnson A.T. Othman M.I. Scott K. Downs C.A. Nguyen T.D. Polansky J.R. Richards J.E. GLC1A mutations point to regions of potential functional importance on the TIGR/MYOC protein.Mol. Vis. 1998; 4: 20Google Scholar) extended their evaluations to a slightly more divergent set of proteins, including a rat latrophilin sequence and a human olfactomedin-related sequence. Subsequently, Kulkarni et al. (6.Kulkarni N. Karavanich C. Atchley W. Anholt R. Characterization and differential expression of a human gene family of olfactomedin-related proteins.Genet. Res. 2000; 76: 41-50Google Scholar) have identified a human olfactomedin gene family of which TIGR/myocilin is one member. The members of this family, designated HOLFA, HOLFB, HOLFC, HOLFD, and HTIGR, are distributed in various tissue types. For example, HOLFB is expressed in pancreatic and prostate tissues, and HOLFC is expressed in cerebellum. In their study, the researchers concentrated their analysis on the olfactomedin domain of the sequences because of extreme differences in sequence lengths among the olfactomedin-related proteins. When the evolutionary relationships between these domains are evaluated, HOLFA and HOLFC appear to be related phylogenetically, as do HOLFB and HOLFD. TIGR/myocilin appears to belong to a separate subgroup. In addition to the human gene family, Kulkarni et al. (6.Kulkarni N. Karavanich C. Atchley W. Anholt R. Characterization and differential expression of a human gene family of olfactomedin-related proteins.Genet. Res. 2000; 76: 41-50Google Scholar) examined 13 other human and non-human sequences, including latrophilins and several ESTs. 1The abbreviations used are: EST, expresses sequence tag; ODC, olfactomedin domain-containing; LR, leucine-rich; LZ, leucine-zipper. 1The abbreviations used are: EST, expresses sequence tag; ODC, olfactomedin domain-containing; LR, leucine-rich; LZ, leucine-zipper. In this study, we have extended the analysis of the human olfactomedin-related gene family. Despite continued interest in the olfactomedin domain-containing (ODC) proteins, the native function has not been determined for any of these proteins. Presumably, sequence elements conserved across long evolutionary times are likely to be essential to the structural or functional success of a protein family (7.McClure M. Vasi T. Fitch W. Comparative analysis of multiple protein-sequence alignment methods.Mol. Biol. Evol. 1994; 11: 571-592Google Scholar). To elucidate potential sequence epitopes important to the native functions of the TIGR sequence, we have attempted to identify a larger, more diverse family of related sequences ( 75% identity) were removed prior to identification of conserved residues using the JalView sequence-editing program (circinus.ebi.ac.uk:6543/jalview/). The human TIGR protein was added back to each segment for comparison after the analysis was complete. Figs. 1 and 2 show the C-terminal and N-terminal domains, respectively, of human TIGR (HTIGR), chicken olfactomedin (COLFA), frog olfactomedin (FOLFA), and bovine latrophilin (BLAT1) aligned with the new sequences identified in our searches. Although most of the sequences span the full length of TIGR and olfactomedin, several are restricted to either terminal end of the alignment. No sequences were identified spanning only the central (i.e. exon 2) region of the TIGR molecule. The newly identified sequences from this work are described in Table I. 3The following sequences were identified by our searches: DOLFA, NCBI accession number AAF48788; JOLHN, NCBI accession number AV670132 and NCBI accession number AV670616; MKIDN, NCBI accession number AI987584; HOLFE, NCBI accession number XP_001313; HOLFF, NCBI accession number CAC17635; HEST4, NCBI accession number AL562289; HEST3, NCBI accession number BF530912; HL-P4, NCBI accession number T23140; ZEST1, NCBI accession number AW115690; ZEST2, NCBI accession number AW154600. Because these searches were completed, the HOLFE sequence has been deleted from GenPept at the request of the submitter; however, a mouse sequence 86% identical to HOLFE is still present with NCBI accession number AAH05485. Fig. 2Alignment of N-terminal regions of HTIGR and representative olfactomedin and latrophilin sequences with newly identified sequences. Residues shaded light gray are predicted α-helices. Residues shaded dark gray are predicted β-sheets. The leucine zipper/leucine-rich regions are highlighted in bold and indicated with z's below the aligned positions. The conserved GXCXXT motif is also shown in bold.View Large Image Figure ViewerDownload (PPT)Table 1ODC family sequences identified from BLAST and Shotgun searchesNameAccession No.Species/descriptionRegion of homologyDOLFAAAF48788CG6867 gene product (Drosophila melanogaster)C-terminalJOLHNAV670132 AV670616OLHNI cell line cDNA library (OLb) Oryzias latipes cDNA clones OLb17.12c and OLb25.05h (composite sequence)N- and C-terminalMKIDNAI987584ul86a03.y1 Sugano mouse kidney mkia Mus musculus cDNA cloneN- and C-terminalHOLFEXP_001313HNOEL-iso protein (Homo sapiens)N- and C-terminalHOLFFCAC17635bA209J19.1.1 (GW112 protein) (Homo sapiens)N- and C-terminalHEST3BF530912NCI_CGAP_Brn67 Homo sapiens cDNA cloneN- and C-terminalHEST4AL562289LTI_NFL003_NBC3 Homo sapiens cDNA cloneC-terminalZEST1AW115690Sugano Kawakami zebrafish DRA Danio rerio cDNA clone 2599775C-terminalZEST2AW154600Sugano Kawakami zebrafish DRA Danio rerio cDNA clone 2639120C-terminalHL-P4T23140Halibut pituitary cDNA T23140C-terminal Open table in a new tab Several of these sequences are interesting in the context of the larger family of ODC proteins. MKIDN, for example, shows significant similarity to the TIGR proteins; however, a large central region of the TIGR molecule is missing in the MKIDN sequence. MKIDN aligns with HTIGR from residue 3 through residue 85 in the exon 1 region. After a 336-residue gap, spanning the leucine zipper and exon 2 regions of TIGR, the alignment continues for the last ∼150 residues of the third exon of TIGR. On the other hand, the homology between TIGR and JOLHN includes regions from each of the three exon regions of TIGR. HEST3 is most similar to HOLFC, and HOLFF is most similar to the frog olfactomedin (FOLFA). The remaining sequences are equally divergent from both the TIGR and olfactomedin sequences but do appear to be evolutionarily related to the group. Consistent with the conclusions of Karavanich and Anholt (2.Karavanich C.A. Anholt R.R. Molecular evolution of olfactomedin.Mol. Biol. Evol. 1998; 15: 718-726Google Scholar) and Kulkarni et al. (6.Kulkarni N. Karavanich C. Atchley W. Anholt R. Characterization and differential expression of a human gene family of olfactomedin-related proteins.Genet. Res. 2000; 76: 41-50Google Scholar), the C-terminal region is well conserved and appears to have evolved slowly, predominantly via point mutations; the N-terminal region, on the other hand, seems to have evolved more quickly. Because of the different apparent rates of evolution between the ends of the molecule, we have analyzed these two regions separately. Fig. 2 shows a ClustalW alignment of a representative set of the N-terminal regions of the sequences of Kulkarni et al. (6.Kulkarni N. Karavanich C. Atchley W. Anholt R. Characterization and differential expression of a human gene family of olfactomedin-related proteins.Genet. Res. 2000; 76: 41-50Google Scholar), olfactomedins, TIGR orthologs, and all of the sequences we have identified in this work containing an N-terminal region. In this region, the similarity among the JOLHN, MKIDN, HOLFE, HOLFF, and HOLFC sequences and the remaining olfactomedin, TIGR sequences is evident. The GXCXXT consensus motif is clear in these sequences, as well as the TIGR and olfactomedin sequences. Also, several of the sequences possess a residual leucine-zipper (LZ) or a leucine-rich (LR) region in their N terminus. Fig. 2 also shows the obvious disparity among the latrophilins (represented by BLAT1), DOLFA, and WOLF2 and the remaining sequences. These proteins lack the GXCXXT motif, as well as a residual LR/LZ region. In our pursuit of distantly related sequences that might be part of a TIGR/olfactomedin superfamily, we searched for homology to the N-terminal region of the sequences shown in Fig. 2. Our congruence analysis using Shotgun did not identify any distantly related sequences with significant similarity to the set other than their LR/LZ regions. In fact, the list of Shotgun hits consisted primarily of sequences with LR or LZ regions. The hits included proteins such as myosin, plectin, kinesin, tropomyosin, and several other coiled-coil sequences. Previous work has shown that this similarity does not appear to be a sufficient basis to infer an evolutionary relationship (1.Green M.L. Do H. Polansky J.R. Nguyen T.D. Klein T.E. Similarities and differences between the TIGR and olfactomedin proteins.Invest. Ophthalmol. Vis. Sci. 2001; 42 (Abstr. 3531): 656Google Scholar). In distantly related proteins, structure is often more highly conserved than sequence (11.Murzin A. How far divergent evolution goes in proteins.Curr. Opin. Struct. Biol. 1998; 8: 380-387Google Scholar). Although proteins sharing 30% sequence identity are expected to exhibit the same fold structure, many proteins with statistically insignificant sequence similarity may also have similar folds (12.Orengo C. Jones D. Thornton J. Protein superfamilies and domain superfolds.Nature. 1994; 372: 631-634Google Scholar, 13.Abagyan R. Batalov S. Do aligned sequences share the same fold?.J. Mol. Biol. 1997; 273: 355-368Google Scholar). Because the coiled-coil structure and LR/LZ domain appeared to be the basis for the similarity between our query sequences and their Shotgun hits, we decided to evaluate our sequences based on their structures. Three-dimensional structural information is not available for any of these sequences; hence we compared the predicted secondary structures for the proteins. Because the composition of a set of sequences can significantly affect a multiple alignment and, hence, the resultant secondary structure prediction, several separate predictions were made for the N-terminal region. The TIGR-like sequences, the olfactomedin-like sequences, and the latrophilin sequences were each submitted separately for secondary structure predictions. Fig. 2 shows a representative sequence from each prediction with the secondary structure elements, helices and β-strands, indicated for each group. The sequences that did not clearly fall into one of these groups were also submitted individually to confirm their predictions were consistent with the larger group. The predicted secondary structures confirm the results of the sequence alignments; DOLFA, WOLF2, and the latrophilins appear to be part of groups separate from that of the olfactomedin- and TIGR-related sequences. All three sequences (DOLFA, WOLF2, and BLAT1) are primarily sheet-like in the N-terminal region. The TIGR- and olfactomedin-related sequences are all primarily helical. Although there are differences in the initiation and termination of the helices and sheets in the separate predictions, the predictions are quite similar for the TIGR- and olfactomedin-related sequences. JOLHN, HOLFE, and HOLFF are also predicted to be predominantly helical in the N-terminal region. We also used Jpred to predict the secondary structure of the C-terminal region. The entire family of ODC sequences aligned by ClustalW was submitted to the Jpred server. Each program predicted that the majority of this domain is composed of β-sheets, with only a few very short helical regions as shown in Fig. 3 for the HTIGR sequence. The figure also includes the N-terminal region of TIGR to highlight the explicit difference in the predicted structures of the two domains. In the C-terminal olfactomedin domain, our analysis included 32 sequences described previously and in the current work. Karavanich and Anholt (2.Karavanich C.A. Anholt R.R. Molecular evolution of olfactomedin.Mol. Biol. Evol. 1998; 15: 718-726Google Scholar) identified five conserved motifs in this domain; however, these motifs were assessed using only four sequences, rat, mouse, and frog olfactomedin and an EST from Caenorhabditis elegans. Our analysis of a larger evolutionarily divergent collection of sequences has enabled us to specify a more stringent set of conserved residues that might be used to recognize more distant members of this group. As discussed previously, we divided the olfactomedin domain into nine segments for our analysis and then removed redundant sequences to <75% identity. The conserved motifs within each of the nine segments are shown in Fig. 4. Segments 2 and 4 are not shown, because they did not contain any residues strongly conserved by identity or by type. Some of the motifs shown in Fig. 4 may appear to include sequences with higher than 75% identity. The <75% sequence identity comparison and the identification of conserved residues were performed on the entire segment, rather than just the conserved portions shown in the figure. For example, in region 1, BLAT1 and BLAT3 are 100% identical (HQSGAWCKDPL); however, the surrounding sequences in segment 1 are more divergent (<75% identical). The exception to this rule is the HTIGR sequence. To maintain a consistent reference sequence throughout the comparison, and because we were interested primarily in the relationship of the other family members to the HTIGR sequence, it was added back to the alignments after the conservation analyses were completed. Five residues within the nine segments of the olfactomedin domain are conserved completely across all of the sequences (excluding those not present in a particular segment). Three of these conservations occur in segment 1 (Gly-268, Trp-270, and Asp-273 in the HTIGR sequence), one occurs in segment 5 (Glu-385), and one occurs in segment 9 (Asn-480). Several of the highlighted motifs were identified by Karavanich and Anholt (2.Karavanich C.A. Anholt R.R. Molecular evolution of olfactomedin.Mol. Biol. Evol. 1998; 15: 718-726Google Scholar) and Nguyen et al. (4.Nguyen T.D. Chen P. Huang W.D. Chen H. Johnson D. Polansky J.R. Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells.J. Biol. Chem. 1998; 273: 6341-6350Google Scholar). The additional evolutionary information included in our divergent alignment helps to clarify the residues that have been conserved across evolutionary time. For instance, the DXDXXXDEGLW motif is not as well conserved across our whole set of ODC sequences as it is among the olfactomedin and TIGR sequences alone. One of the most extensive conserved motifs does not contain any fully conserved residues. The area containing the cysteine shown to be involved in the formation of oligomers in olfactomedin and believed to be involved in dimerization in TIGR (Cys-433; region 7) has no fully conserved residues. This surprising result is primarily because of the presence of two sequences, HEST4 and ZEST2. The ZEST2 sequence is consistent with the conserved motif, with the exception of an arginine in place of the conserved cysteine. Although the HEST4 sequence possesses a cysteine in the conserved position, the surrounding residues do not match the motif. Both HEST4 and ZEST2 are EST sequences and contain nonsense codons upstream of this motif. Future studies should investigate the accuracy of these sequences in this region. Presumably, sequence elements essential to the function or structure of a family of evolutionarily related proteins should be more highly conserved than less essential elements. When functional or structural information is available for a family of proteins, differences among the proteins can be useful for determining which conserved residues are key elements in its function or structure (3.Babbitt P. Gerlt J. Understanding enzyme superfamilies. Chemistry as the fundamental determinant in the evolution of new catalytic activities.J. Biol. Chem. 1997; 272: 30591-30594Google Scholar). Partially conserved or divergent residues, which impart differences in function or structure, may also be identified. Because the normal function and three-dimensional structure of the TIGR and olfactomedin protein families have not been elucidated, another source of information must be found. Rozsa et al. (5.Rozsa F.W. Shimizu S. Lichter P.R. Johnson A.T. Othman M.I. Scott K. Downs C.A. Nguyen T.D. Polansky J.R. Richards J.E. GLC1A mutations point to regions of potential functional importance on the TIGR/MYOC protein.Mol. Vis. 1998; 4: 20Google Scholar) evaluated the effects of mutations on the predicted secondary structure in several TIGR/olfactomedin-related proteins (HTIGR, MTIGR, FOLFA, ROLFA, HOLFA, and RCL2B). The predicted effects of these mutations on the secondary structure of human TIGR provide some insight into the pathogenesis of glaucoma. Likewise, our previous work examined mutations in light of the evolutionary relationship between the TIGR (HTIGR, MTIGR, RTIGR, and BTIGR) and olfactomedin (COLFA, ROLFA, FOLFA, HOLFC, and MOLFA) sequences. Evaluating HTIGR mutations in terms of the conserved sequence elements across a more diverse set of sequences should provide additional understanding of the structural and functional characteristics of the ODC family. The comparison of disease-associated mutations versus non-disease-associated polymorphisms in terms of their degree of conservation as the family has evolved may help to elucidate those residues/motifs that are functionally or structurally essential. We compared 11 polymorphisms and 27 mutations catalogued by several researchers (5.Rozsa F.W. Shimizu S. Lichter P.R. Johnson A.T. Othman M.I. Scott K. Downs C.A. Nguyen T.D. Polansky J.R. Richards J.E. GLC1A mutations point to regions of potential functional importance on the TIGR/MYOC protein.Mol. Vis. 1998; 4: 20Google Scholar, 14.Adam M.F. Belmouden A. Binisti P. Brézin A.P. Valtot F. Béchetoille A. Dascotte J.C. Copin B. Gomez L. Chaventré A. Bach J.F. Garchon H.J. Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma.Hum. Mol. Genet. 1997; 6: 2091-2097Google Scholar, 15.Alward W.L.M. Fingert J.H. Coote M.A. Johnson A.T. Lerner S.F. Junqua D. Durcan F.J. McCartney P.J. Mackey D.A. Sheffield V.C. Stone E.M. Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A).N. Engl. J. Med. 1998; 338: 1022-1027Google Scholar, 16.Angius A. De Gioia E. Loi A. Fossarello M. Sole G. Orzalesi N. Grignolo F. Cao A. Pirastu M. A novel mutation in the GLC1A gene causes juvenile open-angle glaucoma in 4 families from the Italian region of Puglia.Arch. Ophthalmol. 1998; 116: 793-797Google Scholar, 17.Fingert J.H. Ying L. S
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