Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

The human type I collagen mutation database.

Type I collagen is the most abundant and ubiquitously distributed of the collagen family of proteins. It is a heterotrimer comprising two alpha1(I) chains and one alpha2(I) chain which are encoded by the unlinked loci COL1A1 and COL1A2 respectively. Mutations at these loci result primarily in the connective tissue disorders osteogenesis imperfecta and Ehlers-Danlos syndrome types VIIA and VIIB. Two instances of osteoporosis and a single instance of Marfan syndrome are also the result of mutations at these loci. The mutation data are accessible on the world wide web at http://www.le.ac.uk/depts/ge/collagen/collagen.html     

MSEA: detection and quantification of mutation hotspots through mutation set enrichment analysis

Many cancer genes form mutation hotspots that disrupt their functional domains or active sites, leading to gain- or loss-of-function. We propose a mutation set enrichment analysis (MSEA) implemented by two novel methods, MSEA-clust and MSEA-domain, to predict cancer genes based on mutation hotspot patterns. MSEA methods are evaluated by both simulated and real cancer data. We find approximately 51% of the eligible known cancer genes form detectable mutation hotspots. Application of MSEA in eight cancers reveals a total of 82 genes with mutation hotspots, including well-studied cancer genes, known cancer genes re-found in new cancer types, and novel cancer genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0489-9) contains supplementary material, which is available to authorized users.     

Mechanical implications of the domain structure of fiber-forming collagens: comparison of the molecular and fibrillar flexibilities of the alpha1-chains found in types I-III collagen

Fibrillar collagens store, transmit and dissipate elastic energy during tensile deformation. Results of previous studies suggest that the collagen molecule is made up of alternating rigid and flexible domains, and extension of the flexible domains is associated with elastic energy storage. In this study, we model the flexibility of the alpha1-chains found in types I-III collagen molecules and microfibrils in order to understand the molecular basis of elastic energy storage in collagen fibers by analysing the areas under conformational plots for dipeptide sequences. Results of stereochemical modeling suggest that the collagen triple helix is made up of rigid and flexible domains that alternate with periods that are multiples of three amino acid residues. The relative flexibility of dipeptide sequences found in the flexible regions is about a factor of five higher than that found for the flexibility of the rigid regions, and the flexibility of types II and III collagen molecules appears to be higher than that found for the type I collagen molecule. The different collagen alpha1-chains were compared by correlating the flexibilities. The results suggest that the flexibilities of the alpha1-chains of types I and III collagen are more closely related than the flexibilities of the alpha1-chains in types I and II and II and III collagen. The flexible domains found in the alpha1-chains of types I-III collagen were found to be conserved in the microfibril and had periods of about 15 amino acid residues and multiples thereof. The flexibility profiles of types I and II collagen microfibrils were found to be more highly correlated than those for types I and III and II and III. These results suggest that the domain structure of the alpha1-chains found in types I-III collagen is an efficient means for storage of elastic energy during stretching while preserving the triple helical structure of the overall molecule. It is proposed that all collagens that form fibers are designed to act as storage elements for elastic energy. The function of fibers rich in type I collagen is to store and then transmit this energy while fibers rich in types II and III collagen may store and then reflect elastic energy for dissipation through viscous fibrillar slippage. Impaired elastic energy storage by extracellular matrices may lead to cellular damage and changes in signaling by mechanochemical transduction at the extracellular matrix-cell interface.     

Collagens, modifying enzymes and their mutations in humans, flies and worms

Collagens and proteins with collagen-like domains form large superfamilies in various species, and the numbers of known family members are increasing constantly. Vertebrates have at least 27 collagen types with 42 distinct polypeptide chains, >20 additional proteins with collagen-like domains and approximately 20 isoenzymes of various collagen-modifying enzymes. Caenorhabditis elegans has approximately 175 cuticle collagen polypeptides and two basement membrane collagens. Drosophila melanogaster has far fewer collagens than many other species but has approximately 20 polypeptides similar to the catalytic subunits of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. More than 1300 mutations have so far been characterized in 23 of the 42 human collagen genes in various diseases, and many mouse models and C. elegans mutants are also available to analyse the collagen gene family and their modifying enzymes.     

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.     

Type XIII collagen and some other transmembrane collagens contain two separate coiled-coil motifs,which may function as independent oligomerization domains

Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1-3) separated by short non-collagenous domains (NC1-4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an alpha-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen alpha chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule.     

Polycystic kidney disease-like domains of clostridial collagenases and their role in collagen recruitment

Bacterial collagenases exhibit a multimodular domain organization. While the N-terminal collagenase unit harbors the catalytic zinc and suffices to degrade peptidic substrates, collagen substrates come in different types, explaining the requirement for accessory domains such as polycystic kidney disease (PKD)-like domains for efficient catalysis. How the recognition and unfolding of (micro-)fibrillar or triple-helical collagen is accomplished are only poorly understood. Here, we present the crystal structure of the PKD-like domain of collagenase G from Clostridium histolyticum. The β-barrel structure reveals a two-tier architecture, connected by kinked hinge segments. Together with sheet extension as a generic oligomerization mechanism, this explains the cooperativity among accessory domains as well as their adaptivity to varying substrates.     

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.     

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.     

Molecular analysis of collagen binding by the human discoidin domain receptors,DDR1 and DDR2. Identification of collagen binding sites in DDR2

The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.     

Type XII collagen. A large multidomain molecule with partial homology to type IX collagen

Three overlapping cDNAs encoding alpha 1 (XII) collagen have been isolated and sequenced. The DNAs define five sequence domains within the chain. Three domains are nontriple-helical; two are relatively short triple-helical regions. The amino acid sequences of tryptic peptides derived from 16- and 10-kDa pepsin-resistant fragments isolated from tendon extracts are in full agreement with the deduced sequences of the triple-helical regions. Two of the five sequence domains in alpha 1 (XII), one triple-helical and one nontriple-helical, show a high degree of similarity to regions in type IX collagen chains. In addition, examination of seven exons in the alpha 1 (XII) gene shows that the gene is, in part, similar to the structure of type IX collagen genes. Therefore, collagen types IX and XII are partially homologous. The alpha 1 (XII) sequence data predict an asymmetric structure for type XII collagen molecules, fully consistent with the rotary shadowing images. These images show a triple-helical 75-nm tail attached through a central globule to three finger-like structures, each 60 nm long (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156).     

Immunoidentification of type XII collagen in embryonic tissues

We have generated a monoclonal antibody against a synthetic peptide whose sequence was derived from the nucleotide sequence of a cDNA encoding alpha 1(XII) collagen. The antibody, 75d7, has been used to identify the alpha 1(XII) chain on immunoblots of SDS-PAGE tendon extracts as a 220-kD polypeptide, under reducing conditions. Amino-terminal amino acid sequence analysis of an immunopurified cyanogen bromide fragment of type XII collagen from embryonic chick tendons gave a single sequence identical to that predicted from the cDNA, thus confirming that the antibody recognizes the type XII protein. Immunofluorescence studies with the antibody demonstrate that type XII collagen is localized in type I-containing dense connective tissue structures such as tendons, ligaments, perichondrium, and periosteum. With these data, taken together with previous results showing that a portion of the sequence domains of type XII collagen is similar to domains of type IX, a nonfibrillar collagen associated with cross-striated fibrils in cartilage, we suggest that types IX and XII collagens are members of a distinct class of extracellular matrix proteins found in association with quarter-staggered collagen fibrils.     

The cysteine-rich domain of snake venom metalloproteinases is a ligand for von Willebrand factor A domains: role in substrate targeting

Snake venom metalloproteinases (SVMPs) are members of the Reprolysin family of metalloproteinases to which the ADAM (a disintegrin and metalloproteinase) proteins also belong. The disintegrin-like/cysteine-rich domains of the ADAMs have been implicated in their function. In the case of the SVMPs, we hypothesized that these domains could function to target the metalloproteinases to key extracellular matrix proteins or cell surface proteins. Initially we detected interaction of collagen XIV, a fibril-associated collagen with interrupted triple helices containing von Willebrand factor A (VWA) domains, with the PIII SVMP catrocollastatin. Next we investigated whether other VWA domain-containing matrix proteins could support the binding of PIII SVMPs. Using surface plasmon resonance, the PIII SVMP jararhagin and a recombinant cysteine-rich domain from a PIII SVMP were demonstrated to bind to collagen XIV, collagen XII, and matrilins 1, 3, and 4. Jararhagin was shown to cleave these proteins predominantly at sites localized at or near the VWA domains suggesting that it is the VWA domains to which the PIII SVMPs are binding via their cysteine-rich domain. In light of the fact that these extracellular matrix proteins function to stabilize matrix, targeting the SVMPs to these proteins followed by their specific cleavage could promote the destabilization of extracellular matrix and cell-matrix interactions and in the case of capillaries could contribute to their disruption and hemorrhage. Although there is only limited structural homology shared by the cysteine-rich domains of the PIII SVMPs and the ADAMs our results suggest an analogous function for the cysteine-rich domains in certain members of the expanded ADAM family of proteins to target them to VWA domain-containing proteins.     

Type VI collagen of the intervertebral disc. Biochemical and electron-microscopic characterization of the native protein.

The collagen framework of the intervertebral disc contains two major fibril-forming collagens, types I and II. Smaller amounts of other types of collagen are also present. On examination of the nature and distribution of these minor collagens within bovine disc tissue, type VI collagen was found to be unusually abundant. It accounted for about 20% of the total collagen in calf nucleus pulposus, and about 5% in the annulus fibrosus. It was discovered by serially digesting disc tissue with chondroitin ABC lyase and Streptomyces hyaluronidase that native covalent polymers of type VI collagen could be extracted. Electron micrographs of this material prepared by rotary shadowing revealed the characteristic dimensions of tetramers and double tetramers of type VI molecules, with their central rods and terminal globular domains. Molecular-sieve column chromatography on agarose under non-reducing non-denaturing conditions gave a series of protein peaks with molecular sizes equivalent to the tetramer, double tetramer and higher multimers. On SDS/polyacrylamide-gel electrophoresis after disulphide cleavage, these fractions of type VI collagen all showed a main band at Mr 140,000 and four lesser bands between Mr 180,000 and 240,000. On electrophoresis without disulphide cleavage in agarose/2.4% polyacrylamide only dimeric (six chains) and tetrameric (12 chains) forms of type VI molecules were present. The ability to extract all the type VI collagen of the tissue in 4 M-guanidinium chloride, and absence of aldehyde-mediated cross-linking residues on direct analysis, showed that, in contrast with most matrix collagens, type VI collagen does not function as a covalently cross-linked structural polymer.     

Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.     

Preparative procedures and purity assessment of collagen proteins

Collagens represent a large family (25 members identified so far) of closely related proteins. While the preparative procedures for the members that are ubiquitous and present in tissues in large quantities (typically fibre and network forming collagens types I, II, III, IV and V) are well established, the procedures for more recently discovered minor collagen types, namely those possessing large non-collagenous domain(s) in their molecule, are mostly micropreparative and for some collagenous proteins even do not exist. The reason is that the proof of their existence is based on immunochemical staining of tissue slices and nucleic database searching. Methods of preparation and identification of constituting alpha-polypeptide chains as well as collagenous and non-collagenous domains are also reviewed. Methods for revealing non-enzymatic posttranslational modifications (particularly of the fibre forming collagen types) are briefly described as well.