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.     

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.     

Glomerular basement membrane. Identification of a fourth chain, alpha 4, of type IV collagen

The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). M3 was identified as the noncollagenous domain of a fourth chain, alpha 4, of type IV collagen, on the basis of additional sequence data together with previous findings. A comparison of the collagenous-noncollagenous junction regions of alpha 3(IV) and alpha 4(IV) chains with those of classical alpha 1(IV) and alpha 2(IV) chains reveals structural information which provides a potential strategy for molecular cloning of these novel chains. The results further reveal the complexity of electrophoresis patterns of the hexamer and potential ambiguities in using one-dimensional patterns to determine whether molecular defects of collagen IV occur in pathological processes affecting basement membranes.     

Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.     

Characterization of the gamma subunits of the 7S nerve growth factor complex.

The gamma subunits of the 7S nerve growth factor complex (7S NGF) display arginine esteropeptidase activity. By varying the conditions of electrophoresis in acrylamide gel, it has been demonstrated that the gamma-subunit fraction of 7S NGF contains five different proteins, in contrast to the three (gamma1, gamma2, and gamma3) originally described (Smith, A.P., Varon, S. and Shooter, E.M. (1968), Biochemistry 7, 3259-3268); the gamma1 and gamma2 subunits, previously thought to be single species, can each be resolved into two components. The two components of the gamma1 subunit have the same isoelectric point, as do the two components of the gamma2 subunit. The distribution of protein among the two components of each of the gamma1 and gamma2 subunits varied from preparation to preparation. Moreover, a shift in the distribution for the gamma1 subunit was accompanied by a parallel shift for the gamma2 subunit. All of the different gamma proteins have the same molecular weight. On the basis of the molecular weights of the peptide chains of the gamma subunits and of the species which are formed by cross-linking with dimethyl suberimidate, it was concluded, that both the gamma1 and gamma2 subunits contain one species with two peptide chains and another with three peptide chains, while the gamma3 subunit is a single species with three peptide chains. The results also suggest that two of the chains in the three-chain species are derived, by proteolytic cleavage, from the larger chain in the two-chain species.     

Biosynthesis and in vitro translation of type IV procollagens

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains alpha 1(IV) and alpha 2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of alpha, alpha'-bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis.     

OsPAA2, a distinct alpha 1 subunit gene for the 20S proteasome in rice (Oryza sativa L.)

The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins and other diverse biological functions. The 20S proteasome is constituted of 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and the two inner rings are composed of seven different alpha and beta type subunits, respectively, giving an alpha 7 beta 7 beta 7 alpha 7 structure. We previously reported the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all the subfamilies from monocot. In this study, a distinct cDNA sequence encoding the alpha1 subunit, OsPAA2, was identified. The amino acid sequence similarity between the two rice alpha1 subunits was as low as 59.6%, contrasting with those between paralogs of Arabidopsis proteasome subunit genes. The expression pattern of the OsPAA2 gene was different from that of another alpha1 gene, OsPAA1. These data suggest that OsPAA2 might play a distinct role from that of OsPAA1 in the 20S proteasome complex.     

Comparison of the crystal structures of L2 and L8S8 Rubisco suggests a functional role for the small subunit.

Comparison of the crystal structures of the L2 and L8S8 forms of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum and spinach respectively, reveals a remarkable similarity in the overall architecture of the L2 building blocks in the two enzymes. Within the L subunits, no large conformational differences such as domain-domain rotations were found. In spite of a somewhat different packing of the L subunits in the L2 dimer, the active sites of the two enzymes are highly conserved. Significant local conformational differences are, however, observed for the C-terminal part of the polypeptide chains as well as for loop 7, helix alpha 7, loop 8 and helix alpha 8 in the barrel domain. The small subunit forms extensive interactions with one of these alpha helices, alpha 8, in the spinach L8S8 enzyme. The loops are at the active site and one of them forms a phosphate binding site for the substrate. We suggest that the small subunit modulates substrate binding and, possibly, the carboxylation/oxygenation ratio by inducing conformational changes in the active site through interactions distant from this site.     

Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha1 subunit

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.     

Complete primary structure of the triple-helical region and the carboxyl-terminal domain of a new type IV collagen chain, alpha 5(IV)

We have isolated and characterized overlapping cDNA clones which code for a previously unidentified human collagen chain. Although the cDNA-derived primary structure of this new polypeptide is very similar to the basement membrane collagen alpha 1(IV) and alpha 2(IV) chains, the carboxyl-terminal collagenous/non-collagenous junction sequence does not correspond to the junction sequence in either of the newly described alpha 3(IV) or alpha 4(IV) chains (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B. G. (1987) J. Biol. Chem. 262, 7874-7877). Thus the protein presented here has been designated the alpha 5 chain of type IV collagen. Four clones encode an open reading frame of 1602 amino acids that cover about 95% of the entire chain including half of the amino-terminal 7S domain and all of the central triple-helical region and carboxyl-terminal NC1 domain. The collagenous region of the alpha 5(IV) chain contains 22 interruptions which are in most cases identical in distribution to those in both the alpha 1(IV) and alpha 2(IV) chains. Despite the relatively low degree of conservation among the amino acids in the triple-helical region of the three type IV collagen chains, analysis of the sequences clearly showed that alpha 5(IV) is more related to alpha 1(IV) than to alpha 2(IV). This similarity between the alpha 5(IV) and alpha 1(IV) chains is particularly evident in the NC1 domains where the two polypeptides are 83% identical in contrast to the alpha 5(IV) and alpha 2(IV) identity of 63%. In addition to greatly increasing the complexity of basement membranes, the alpha 5 chain of type IV collagen may be responsible for specialized functions of some of these extracellular matrices. In this regard, it is important to note that we have recently assigned the alpha 5(IV) gene to the region of the X chromosome containing the locus for a familial type of hereditary nephritis known as Alport syndrome (Myers, J.C., Jones, T.A., Pohjalainen, E.-R., Kadri, A.S., Goddard, A.D., Sheer, D., Solomon, E., and Pihlajaniemi, T. (1990) Am. J. Hum. Genet. 46, 1024-1033). Consequently, the newly discovered alpha 5(IV) collagen chain may have a critical role in inherited diseases of connective tissue.     

Subunit interactions involved in the assembly of pyridine nucleotide transhydrogenase in the membranes of Escherichia coli.

The pyridine nucleotide transhydrogenase (PNT) of Escherichia coli consists of two different subunits (alpha and beta) and assembles as a tetramer (alpha 2 beta 2) in the inner membrane. The pnt genes from E. coli have been cloned on a multicopy plasmid resulting in high level expression of the enzyme activity. We have studied the influence of the different segments of the polypeptide chains of the alpha and beta subunits on the assembly and function of the enzyme by constructing a series of deletion mutants for both of the subunits. Our results show that the assembly of the beta subunit is contingent upon the insertion of the alpha subunit into the membrane, while the alpha subunit can assemble independently of the beta subunit. All deletions constructed for the cytosolic portion of the alpha subunit gave no incorporation of the alpha subunit and, as a consequence, of the beta subunit, also. Of the four membrane-spanning regions of the alpha subunit, the last two were indispensable, while the deletion of the first two still allowed the association of alpha as well as of the beta subunit with the membrane. However, the enzyme was not functional. The two subunits were also loosely associated as mild detergent treatment released them from the membrane in contrast with the wild-type enzyme. Deletions within the beta subunit had little effect on the assembly of the alpha subunit, although less was incorporated. All deletions involving the cytosolic portion of the beta subunit resulted in loss of incorporation into the membrane. Of the eight membrane-spanning regions of the beta subunit, the deletion of regions 2-3, 2-4, 2-6, and 2-7 yielded significant association of both the subunits with the membrane. However, none of these mutants assembled a functional enzyme, and again the two subunits were loosely associated with the membrane. Based on the stringent requirement of the cytosolic portions of alpha and beta subunits for assembly, a model is proposed that suggests interactions between these two regions must occur prior to assembly.     

Structural heterogeneity of the noncollagenous domain of basement membrane collagen

The noncollagenous domain of collagen from three different basement membranes of bovine origin (glomerular, lens capsule, and placental) was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. In each case the domain existed as a hexamer comprised of four distinct subunits (alpha 1 (IV) NC1, alpha 2 (IV) NC1, M2*, and M3). Each subunit exists in both monomeric and dimeric (disulfide-cross-linked) forms. Certain dimers also exist which contain nonreducible cross-links. The hexamers from the three membranes differ with respect to stoichiometry of subunits and subunit isoforms and to the degree of cross-linking of monomers into dimers. The minor subunits, M2* and M3, vary in quantity over a 20-fold range relative to the major ones among the three hexamers. The results indicate that: 1) at least two populations of triple-helical collagen molecules, differing in chain composition, exist in each membrane and that their relative proportions are tissue-specific; and 2) the chemical nature of the noncollagenous domain of these populations is tissue-specific with regard to subunit isoforms and relative proportion of reducible and nonreducible cross-links in dimers. A novel structural feature of the noncollagenous domain of basement membrane collagen was also evinced from these studies. Namely, that each of the four monomeric subunits exists in charge isoforms.     

Immunological and chemical characterization of rat liver cytochrome c oxidase

Rat liver cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 different polypeptide chains. Specific antisera against the holoenzyme and against purified subunits IV and VIII were used to characterize the enzyme complex. The antiserum against subunit IV precipitates from sodium dodecyl sulfate-dissociated mitochondria only subunit IV and from Triton X-100-dissolved mitochondria all 12 polypeptide chains, indicating their integral location within the enzyme complex. Different antisera against the holoenzyme only precipitate subunits IV, V and VIb from sodium dodecyl sulfate-dissociated mitochondria, suggesting the location of these subunits on the surface layer of the complex. Subunit VIII is thought to be located within the complex, since a specific antiserum does not precipitate the complex. The amino acid composition of all 12 protein subunits is different, thus excluding their origin from proteolytic degradation. The proteolytic degradation of subunit IV into IV during isolation of the enzyme was corroborated by the very similar amino acid composition of both proteins.     

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.     

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.     

Bovine glomerular basement membrane. Location and structure of the asparagine-linked oligosaccharide units and their potential role in the assembly of the 7 S collagen IV tetramer

Collagen IV contains an amino-terminal tetramerization domain (7 S) that is involved in aggregation and cross-linking as part of the process of self-assembly of the collagen IV matrix of basement membranes. We determined the structure and location of the Asn-linked oligosaccharides of the 7 S tetramer. Two glycopeptides, GP-1 and GP-2, were isolated from tryptic digests of the 7 S tetramer and were characterized. GP-1 and GP-2 are derived from the alpha 1(IV) chain and the alpha 2(IV) chain, respectively. Each glycopeptide contained one sequence, -Asn-Xaa-Thr-, which was shown to be N-glycosylated at Asn, corresponding to position 126 of the alpha 1 chains and 138 of the alpha 2 chain. 1H NMR spectroscopic analysis of the oligosaccharide is a biantennary N-acetyllactosamine type of N-linked oligosaccharide with a broad heterogeneity in the presence of the sugar residues at their nonreducing termini as indicated. [formula: see text] The location of the Asn-linked oligosaccharide units and Hyl-linked disaccharide units and their orientation with respect to the surface of the triple helix were calculated using two models. We conclude that both units are important determinants in the assembly of the 7 S tetramer.     

Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.     

Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice

Type IV collagen includes six genetically distinct polypeptides named alpha1(IV) through alpha6(IV). These isoforms are speculated to organize themselves into unique networks providing mammalian basement membranes specificity and inequality. Recent studies using bovine and human glomerular and testis basement membranes have shown that unique networks of collagen comprising either alpha1 and alpha2 chains or alpha3, alpha4, and alpha5 chains can be identified. These studies have suggested that assembly of alpha5 chain into type IV collagen network is dependent on alpha3 expression where both chains are normally present in the tissue. In the present study, we show that in the lens and inner ear of normal mice, expression of alpha1, alpha2, alpha3, alpha4, and alpha5 chains of type IV collagen can be detected using alpha chain-specific antibodies. In the alpha3(IV) collagen-deficient mice, only the expression of alpha1, alpha2, and alpha5 chains of type IV collagen was detectable. The non-collagenous 1 domain of alpha5 chain was associated with alpha1 in the non-collagenous 1 domain hexamer structure, suggesting that network incorporation of alpha5 is possible in the absence of the alpha3 chain in these tissues. The present study proves that expression of alpha5 is not dependent on the expression of alpha3 chain in these tissues and that alpha5 chain can assemble into basement membranes in the absence of alpha3 chain. These findings support the notion that type IV collagen assembly may be regulated by tissue-specific factors.     

Type VI collagen is composed of a 200 kd subunit and two 140 kd subunits.

We have isolated type VI collagen, a transformation-sensitive glycoprotein of the extracellular matrix, in an intact, disulfide-bonded form. The protein contains a 200 kd subunit and two different 140 kd subunits in a stoichiometric ratio. Based on the amount of hydroxyproline and hydroxylysine, the sensitivity to bacterial collagenase and the cross-reactivity with antibodies to pepsin-extracted type VI collagen, we have identified the 200 kd subunit as the alpha 3(VI) chain and the two 140 kd subunits as the alpha 1(VI) and alpha 2(VI) chains. The alpha 3(VI) chain is synthesized by cells in culture as a precursor of 260 kd, while no precursor form of the other two chains could be detected.