Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

The pro alpha 2(V) collagen gene is evolutionarily related to the major fibrillar-forming collagens.

A number of overlapping cDNA clones, covering 5.2 kb of sequences which code for the human pro alpha 2(V) collagen chain, have been isolated. Analysis of the structural data have indicated a close evolutionary kinship between the pro alpha 2(V) chain and the major fibrillar collagen types. Isolation and analysis of an 8 kb genomic fragment has further supported this notion by revealing a homologous arrangement of nine triple-helical domain exons. These studies have therefore provided conclusive evidence which categorizes the Type V collagen as a member of the Group 1 molecules, or fibrillar-forming collagens.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.     

Identification of the cartilage alpha 1(XI) chain in type V collagen from bovine bone

Type V collagen prepared from bovine bone was resolved into three distinct alpha-chains by high performance liquid chromatography and gel electrophoresis. Peptide mapping established two chains as alpha 1(V) and alpha 2(V) as expected and the third as the cartilage alpha 1(XI) chain (previously thought to be unique to cartilage). In adult bone, the type V collagen fraction was richer in alpha 1(XI) chains than in fetal bone (about 1/3 of the chains in the adult). How these polypeptides are organized into native molecules is not yet clear, though the stoichiometry suggests cross-type heterotrimers between the type V and XI chains.     

Distinct recognition of collagen subtypes by alpha(1)beta(1) and alpha(2)beta(1) integrins. Alpha(1)beta(1) mediates cell adhesion to type XIII collagen

Two integrin-type collagen receptors, alpha(1)beta(1) and alpha(2)beta(1), are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either alpha(1) or alpha(2) integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing alpha(1)beta(1) integrin could spread on collagen types I, III, IV, and V but not on type II, while alpha(2)beta(1) integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction with the integrins has not been previously studied. CHO-alpha1beta1 cells could spread on human recombinant type XIII collagen, unlike CHO-alpha2beta1 cells. Integrins alpha(1)beta(1) and alpha(2)beta(1) recognize collagens with the specific alphaI domains. The alpha(1)I and alpha(2)I domains were produced as recombinant proteins, labeled with europium and used in a sensitive solid-phase binding assay based on time-resolved fluorescence. alpha(1)I domain, unlike the alpha(2)I domain, could attach to type XIII collagen. The results indicate, that alpha(1)beta(1) and alpha(2)beta(1) have different ligand binding specificity. Distinct recognition of different collagen subtypes by the alphaI domains can partially explain the differences seen in cell spreading. However, despite the fact that CHO-alpha1beta1 cells could not spread on type II collagen alpha(1)I domain could bind to this collagen type. Thus, the cell spreading on collagens may also be regulated by factors other than the integrins.     

The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1-F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C-terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N-terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2-alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).     

Complete primary structure of human collagen alpha 1 (V) chain

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.     

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.     

Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.     

The complete primary structure of mouse alpha 2(IV) collagen. Alignment with mouse alpha 1(IV) collagen

We have determined the nucleotide and amino acid sequences of mouse alpha 2(IV) collagen which is 1707 amino acids long. The primary structure includes a putative 28-residue signal peptide and contains three distinct domains: 1) the 7 S domain (residues 29-171), which contains 5 cysteine and 8 lysine residues, is involved in the cross-linking and assembly of four collagen IV molecules; 2) the triple-helical domain (residues 172-1480), which has 24 sequence interruptions in the Gly-X-Y repeat up to 24 residues in length; and 3) the NC1 domain (residues 1481-1707), which is involved in the end-to-end assembly of collagen IV and is the most highly conserved domain of the protein. Alignment of the primary structure of the alpha 2(IV) chain with that of the alpha 1(IV) chain reported in the accompanying paper (Muthukumaran, G., Blumberg, B., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6310-6317) suggests that a heterotrimeric collagen IV molecule contains 26 imperfections in the triple-helical domain. The proposed alignment is consistent with the physical data on the length and flexibility of collagen IV.     

Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3)

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.     

The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.

Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.