Phenotyping of pig α1B-glycoprotein (PO2) and haemopexin by 1D polyacrylamide gel electrophoresis and immunoblotting

Summary. Described is an alternative procedure for the phenotyping of pig α₁B-glycoprotein (PO2) and haemopexin. The procedure is based on the separation of serum samples by horizontal polyacrylamide gel electrophoresis, passive blotting onto a nitrocellulose (NC) sheet, and immunochemical detection using a mixture of a primary antibody (rabbit anti-pig α₁B or anti-pig haemopexin) and a peroxidase-labelled secondary antibody. Several NC copies can be obtained from a single gel and these can be developed with different monospecific antisera.     

Point mutations of single amino acids abolish ability of alpha3 NC1 domain to elicit experimental autoimmune glomerulonephritis in rats

We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins. We now examine a more limited amino-terminal region of alpha3(IV) non-collagenous domain (NC1) and the impact of single amino acid (AA) mutations of this region on glomerulonephritis induction. Rats were immunized with collagenase-solubilized glomerular basement membrane (csGBM), D3, an alpha1(IV)NC1 chimeric protein with 69 AA of alpha3(IV)NC1 (binds Goodpasture sera), D4, the D3 construct shortened by 4 AA (non-binding), P9, P10, single AA mutants (non-binding), and S2, alpha1(IV)NC1 with 9 AA of alpha3(IV)NC1 (binding). All rats immunized with csGBM and S2 and 50% of D3 rats developed glomerulonephritis. csGBM rats had intense GBM-bound IgG deposits, but S2 and D3 rats had minimal deposits. None of the D4, P9, or P10 rats developed glomerulonephritis. Lymphocytes from nephritic rats proliferated with csGBM, S2, and D3, but not with D4, P9, or P10. Discrete segments of alpha3(IV)NC1 within the alpha1(IV)NC1 backbone can induce glomerulonephritis. Single AA mutations within that epitope render the antigen unresponsive to Goodpasture sera and incapable of inducing glomerulonephritis. These studies support the concordance of glomerulonephritis inductivity and Goodpasture serum binding. Further, they define a critical limited AA sequence within alpha3(IV)NC1 of nine or fewer AA, which confers nephritogenicity to the nonnephritogenic alpha1(IV)NC1 without in vivo antibody binding. This region may be a T-cell epitope responsible for induction of glomerulonephritis in this model in rats and Goodpasture syndrome in man.     

Anti-pig antibody adsorption efficacy of {alpha}-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 {beta}1, 6-N-acetylglucosaminyltransferase expression

We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha1,3 galactosyl- and core 2 beta1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.     

The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes

Type IV collagen, the major component of basement membranes (BMs), is a family of six homologous chains (alpha1-alpha6) that have a tissue-specific distribution. The chains assemble into supramolecular networks that differ in the chain composition. In this study, a novel network was identified and characterized in the smooth muscle BMs of aorta and bladder. The noncollagenous (NC1) hexamers solubilized by collagenase digestion were fractionated by affinity chromatography using monoclonal antibodies against the alpha5 and alpha6 NC1 domains and then characterized by two-dimensional gel electrophoresis and Western blotting. Both BMs were found to contain a novel alpha1.alpha2.alpha5.alpha6 network besides the classical alpha1.alpha2 network. The alpha1.alpha2.alpha5.alpha6 network represents a new arrangement in which a protomer (triple-helical isoform) containing the alpha5 and alpha6 chains is linked through NC1-NC1 interactions to an adjoining protomer composed of the alpha1 and alpha2 chains. Re-association studies revealed that the NC1 domains contain recognition sequences sufficient to encode the assembly of both networks. These findings, together with previous ones, indicate that the six chains of type IV collagen are distributed in three major networks (alpha1.alpha2, alpha3.alpha4.alpha5, and alpha1.alpha2.alpha5.alpha6) whose chain composition is encoded by the NC1 domains. The existence of the alpha1.alpha2.alpha5.alpha6 network provides a molecular explanation for the concomitant loss of alpha5 and alpha6 chains from the BMs of patients with X-linked Alport's syndrome.     

The alloantigenic sites of alpha3alpha4alpha5(IV) collagen: pathogenic X-linked alport alloantibodies target two accessible conformational epitopes in the alpha5NC1 domain

Anti-glomerular basement membrane (GBM) antibody nephritis is caused by an autoimmune or alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen. Some patients with X-linked Alport syndrome (XLAS) develop post-transplant nephritis mediated by pathogenic anti-GBM alloantibodies to collagen IV chains present in the renal allograft but absent from the tissues of the patient. In this work, the epitopes targeted by alloantibodies from these patients were identified and characterized. All XLAS alloantibodies recognized conformational epitopes in the NC1 domain of alpha5(IV) collagen, which were mapped using chimeric alpha1/alpha5 NC1 domains expressed in mammalian cells. Allograft-eluted alloantibodies mainly targeted two conformational alloepitopes mapping to alpha5NC1 residues 1-45 and 114-168. These regions also encompassed the major epitopes of circulating XLAS alloantibodies, which in some patients additionally targeted alpha5NC1 residues 169-229. Both kidney-eluted and circulating alloantibodies to alpha5NC1 distinctively targeted epitopes accessible in the alpha3alpha4alpha5NC1 hexamers of human GBM, unlike anti-GBM autoantibodies, which targeted sequestered alpha3NC1 epitopes. The results identify two immunodominant alpha5NC1 epitopes as major alloantigenic sites of alpha3alpha4alpha5(IV) collagen specifically implicated in the pathogenesis of post-transplant nephritis in XLAS patients. The contrast between the accessibility of these alloepitopes and the crypticity of autoepitopes indicates that distinct molecular forms of antigen may initiate the immunopathogenic processes in the two forms of anti-GBM disease.     

Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition

NC1 subunits were purified from gel filtration pools of acid-extracted, collagenase-digested human glomerular basement membranes (hGBM). This methodology, which enriches 28-kDa monomers (M28) in the total digest, allowed purification of these monomers and 24-kDa (M24) and 26-kDa (M26) monomers free from dimers. Reactivity of these subunits with Goodpasture autoantibodies using immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional nonequilibrium pH gradient electrophoresis gels showed strong reactivity with the purified M28 subunits. Inhibition enzyme-linked immunosorbent assay, used to quantitate the reactivity of the purified NC1 subunits, indicated that M28 had a greater than 10-fold increase in ability to inhibit binding to NC1 than NC1 itself. Comparison of hGBM NC1 components were made with those obtained from collagenase digests of salt and acid-extracted bovine and sheep GBM and Englebreth-Holm-Swarm tumor similarly purified by gel filtration and reverse-phase high performance liquid chromatography. Two-dimensional gel analysis of these NC1 isolates revealed absence of the very cationic M28 monomers. Reactivity with antibodies eluted from diseased kidneys of sheep immunized with hGBM (Steblay nephritis) was compared with Goodpasture autoantibody reactivity by immunoblotting two-dimensional gels of hGBM NC1. We conclude that a very cationic M28 monomer (M28 ) found only in hGBM is the probable target in Goodpasture syndrome, that the epitope is present on most NC1 components from extracted and unextracted hGBM, and is exposed by urea denaturation which is enhanced by acid treatment. A weakly cationic M28 monomer (M28+) is present in GBM from other species and is the probable target in Steblay nephritis. Differential recognition of the two M28 components by these antibodies points to different genetic origins or possibly distinct post-translational modifications for these components. This is supported by their presence or absence in different species and tissues, as well as biochemical differences from the M24/26 monomers which presumably are derived from alpha 1(IV) and alpha 2(IV) collagen chains.     

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.     

Crystal structure of the collagen alpha1(VIII) NC1 trimer.

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.     

Alpha 2 beta 1 integrin mediates dermal fibroblast attachment to type VII collagen via a 158-amino-acid segment of the NC1 domain.

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the alpha2 or beta1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202-360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell-matrix interaction is mediated by the alpha2beta1 integrin and is RGD independent.     

Isolation of the haemopexin-haem receptor from pig liver cells

Isolated pig liver plasma membranes interact specifically with the haemopexin-haem complex (Kd 4.4 X 10(-7) M). Affinity chromatography was used to isolate a membrane component which binds this complex with high affinity. Pig serum haemopexin was first isolated by affinity chromatography on haemin-Sepharose followed by HPLC gel filtration. Liver membranes solubilized with Triton X-100 were incubated with haemin-Sepharose saturated with haemopexin, and as a control, with affinity gel lacking haemopexin. SDS-poly-acrylamide gel electrophoresis of the eluted protein indicated that from the haemin-Sepharose emerglow-molecular-mass haemin-binding proteins whereas the eluate from haemopexin-haemin-Sepharose contained an additional 71 kDa protein, which did not bind free haemin. This protein appears to represent the haemopexin-haem receptor or a part of it. Haem from the haemopexin complex, as also free haemin, was accepted by a binder in the plasma membrane, which in gel filtration behaved like an 80 kDa molecule. This component probably represents a second functional subunit of the haemopexin-haem receptor.     

The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane.

Type IV collagen, which has long been assumed to contain two alpha 1(IV) and one alpha 2(IV) chains, also contains alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. Stoichiometry of collagenous alpha(IV) chains differs among tissues, suggesting the existence of subclasses of type IV collagen, each with a unique chain composition. This study seeks to define, by characterization of subunit compositions of NC1 domain populations, the structural organization of type IV collagen from bovine glomerular basement membrane. NC1 hexamers from type IV collagen were separated on two affinity chromatography columns, one containing monoclonal antibodies to the alpha 3 chain, and another, to the alpha 1 chain. SDS-polyacrylamide gel electrophoresis, immunoblotting, reversed phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay identified three NC1 hexamer populations: 1) a hexamer composed of (alpha 1)2 and (alpha 2)2 homodimers; 2) a hexamer composed of (alpha 3)2 and (alpha 4)2 homodimers; 3) a hexamer containing all four alpha chains connected in heterodimers, alpha 1-alpha 3 and alpha 2-alpha 4. Results suggest that there are two distinct type IV collagen molecules, one composed of alpha 1(IV) and alpha 2(IV) chains and another composed of alpha 3(IV) and alpha 4(IV) chains. Furthermore, polymerization occurs between molecules with the same chain composition and between molecules with different chain composition. Moreover, crosslinking between different alpha chains is restricted, thus limiting the number of possible macromolecular structures.     

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Peptide-specific antibodies employed in determining the interspecies immunological cross-reactivity of haemopexin

1. Antibodies were raised in rabbits against nine peptides analogous to sequences of the human serum beta-glycoprotein haemopexin, and seven peptides were very antigenic. 2. One of these affinity-purified peptide-specific antibodies interacted with a highly conserved sequence of the haemopexin of five of the seven species tested. 3. Another antibody bound pig haemopexin even better than human haemopexin. 4. The overall, arbitrarily assessed, immunological cross-reactivity between the haemopexin of human and other species follows the order: rabbit greater than mouse greater than chicken greater than pig greater than rat greater than cow.     

Additional studies of sheep haemopexin: genetic control, frequencies and postnatal development

This study presents evidence that sheep haemopexin phenotypes are genetically controlled by three alleles, HpxA, HpxB1 and HpxB2, of a single autosomal locus. Frequencies of two alleles, HpxA and HpxB (HpxB encompasses two isoalleles, HpxB1 and HpxB2), were studied in eight sheep breeds in Czechoslovakia. The frequency of the HpxA allele was highest (ranging from 0.81 in Merino to 1.0 in East Friesian sheep). Qualitative and quantitative changes in haemopexin during postnatal development were studied by starch gel electrophoresis and rocket immunoelectrophoresis respectively. In electrophoresis, 1- or 2-day-old lambs had two very weak zones corresponding in mobility to two slower zones of adult animals. Later, the third more anodic zone appeared and gradually increased in intensity. In 1-month-old lambs the patterns were practically identical with those of adult animals. Using rocket immunoelectrophoresis, the level of haemopexin shortly after birth was practically zero. It rose sharply till the sixth day of life; then the level continued to rise slowly till about 1 month of age. The mean haemopexin level in adult sheep was 64.5 +/- 18.26 (SD) mg/100ml serum, ranging from 30.5 to 116.5 mg/100ml.     

The goodpasture autoantigen. Identification of multiple cryptic epitopes on the NC1 domain of the alpha3(IV) collagen chain

Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the alpha3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the alpha3(IV) chain as residues 17-31 (E(A)) and 127-141 (E(B)) (Netzer, K.-O. et al. (1999) J. Biol. Chem. 274, 11267-11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the E(A) and/or the E(B) region. Four subpopulations of GP antibodies with distinct epitope specificity for the alpha3(IV)NC1 domain were thus separated and characterized. They were designated GP(A), GP(B), GP(AB), and GP(X), to reflect their reactivity with E(A) only, E(B) only, both regions, and neither, respectively. Hence, regions E(A) and E(B) encompass critical amino acids that constitute three distinct epitopes for GP(A), GP(B), and GP(AB) antibodies, respectively, whereas the epitope for GP(X) antibodies is located in a different unknown region. The GP(A) antibodies were consistently immunodominant, accounting for 60-65% of the total immunoreactivity to alpha3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions E(A) and E(B) are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract alpha3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease.     

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.     

Goodpasture disease. Characterization of a single conformational epitope as the target of pathogenic autoantibodies.

Goodpasture disease is a prototype autoimmune disease characterized by the formation of autoantibodies against the heterotrimeric basement membrane collagen type IV, which causes a rapidly progressive glomerulonephritis. The pathogenic antibody response is directed to the non-collagenous (NC1) domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1), but not to the homologous region of the alpha1(IV)NC1. To identify the conformation-dependent immunodominant epitope on the alpha3(IV)NC1, a variety of recombinant NC1 domains were constructed by replacing single residues of alpha3(IV) with the corresponding amino acids from the nonreactive alpha1(IV) chain. Replacement mutations were identified that completely destroyed the Goodpasture epitope in the alpha3(IV) chain. Based on the identification of these critical positions, the epitope was finally reconstructed within the frame of the alpha1(IV) chain. The substitution of nine discontinuous positions in the alpha1(IV)NC1 with amino acid residues from the alpha3 chain resulted in a recombinant construct that was recognized by all patients' sera (n = 20) but by none of the sera from healthy controls (n = 10). This provides, for the first time, the molecular characterization of a single immunodominant conformational epitope recognized by pathogenic autoantibodies in a human autoimmune disease, representing the basis for the development of new epitope-specific strategies in the treatment of Goodpasture disease.     

Identification and analysis of collagen alpha 1(XXI), a novel member of the FACIT collagen family.

The FACIT collagens bind to the surface of collagen fibrils linking them with other matrix molecules. Bioinformatics analysis of cDNA clone DKFZp564B052 showed that it resembled the FACIT collagens and was therefore designated collagen alpha 1(XXI). Phylogenetic analyses of the N-terminal NC3 domains of alpha 1(XXI) and other FACIT collagens showed that (i) alpha 1(XXI) clustered with the FACIT collagens; (ii) collagen alpha 1(XXI) arose before the divergence of alpha 1(XII), alpha 1(XIV) and alpha 1(XX); (iii) collagen alpha 1(XIV) derived from the C-terminal region of the NC3 domain of a collagen alpha 1(XII)-like molecule; and (iv) collagen alpha 1(XX) derived from a collagen alpha 1(XIV)-like molecule. This study provides a framework for the evolution of the FACIT collagens which will be of value in linking NC3 domains with their functions.