Comparative analysis of the noncollagenous NC1 domain of type IV collagen: Identification of structural features important for assembly, function, and pathogenesis

Abstract

Type IV collagen al-a6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NCl domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, a1-like and a2-like, and suggested that vertebrate a3/a4 sequences evolved before a l / a 2 and a5/a6. About one fifth of NCl residues were identified to confer either the a 1 or a 2 group-specificity. These residues accumulate opposite charge in subdomain B of a1 (positive) and a 2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic P-strands and one short a-helix with a significant hydrophobic moment. The existence of opposite charges in the a-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NCl to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NCl preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function.

Hydrophobic Amino Acid Residues Are Critical for the Immunodominant Epitope of the Goodpasture Autoantigen

“…Since a quantitative data analysis was not reported in that study, it is difficult to directly compare their findings with ours. A very recent paper from the same group (24) quantitatively analyzed the role of four ␣3 residues in the E A region and found Val 27 to be critical for GP antibody binding (similar to our findings), whereas Thr 17 , Ala 18 , and Glu 24 had a moderate effect. Some of the discrepancies may be explained by the different ␣1/␣3 chimeras used as template for the homolog-scanning mutagenesis.…”

“…The ␣3(IV) NC1 sequence was analyzed with the program Epiplot, which calculates and plots flexibility, hydrophilicity, and antigenicity profiles using 13 different scales, chosen as those yielding the best predictions on proteins whose antigenic structures are known (20). None of 27 , and Pro 28 are critical for the GP A epitope (diagonal lines). One or more of these GP A residues are buried within the NC1 hexamer by hydrophobic interactions with residues of other NC1 domains, rendering the epitope cryptic and inaccessible for antibody binding (left).…”

“…That the E A region is a site of interaction between ␣3 and the other NC1 domains in the ␣3⅐␣4⅐␣5 hexamer is deduced from its cryptic nature of the GP A epitope. That the E A region may also be responsible for the specificity of interaction is suggested by the high sequence divergence of this region among the six NC1 domains (27). The E A region is also distinguished by the highest number of non-conservative amino acid substitutions.…”

Mammalian collagen IV

“…The presence of both reducible and nonreducible NC1 dimers have been repeatedly reported in different tissues such as kidney, placenta, aorta, and mouse Engelbreth-Holm-Swarm tumor Langeveld et al, 1988;Siebold et al, 1988;Weber et al, 1984). Such dimers have long been assumed to be disulfide crosslinked monomers formed through a reshuffling mechanism of disulfide bonds during network formation (Netzer et al, 1998b;Siebold et al, 1988). However, the relative amount of nonreducible dimers isolated from different tissues varies significantly, for example 70% in BM of placenta but only 30% in BM of lens capsule Vanacore et al, 2004).…”

Gelatinase-mediated migration and invasion of cancer cells

“…The C-terminal fragment of the progollagen C-terminal proteinase enhancer protein was purified from human brain tumor cells due to its MMP-inhibitory activity, but it is a less potent inhibitor than the TIMPs (Mott et al, 2000). The noncollagenous NC1 domains of collagen type IV are another protein domains with structural similarities to TIMPs (Netzer et al, 1998). Among the NC1 domains of collagen type IV, the α3 chain NC1 domain is the most potent inhibitor of angiogenesis and tumor growth (Petitclerc et al, 2000).…”