Collagen Recognition Research Articles

Structure, stability and interactions of type I collagen with GLY349-CYS substitution in $alpha;1(I) chain in a murine Osteogenesis Imperfecta model

Here we report the structural and functional studies of collagen from the Brtl mouse, a heterozygous knock-in model for Osteogenesis Imperfecta, which has a G349C substitution introduced in one col1a1 allele. We observed that 25±5% of α1(I) chains in different tissues and in different extracts from matrix deposited by cultured cells were S–S-linked mutant dimers. Apparently mutant and normal molecules are equally well incorporated into the matrix and they form mature covalent crosslinks with the same efficiency. We found different extents of post-translational overmodification of mutant molecules in different tissues, but we found no consistent differences between lethal and non-lethal animals. We did not detect any changes in the thermal stability or rate of thermal denaturation of mutant collagen. We also did not detect any changes in collagen–collagen recognition and interactions except for disruption of quasi-crystalline lateral packing of molecules in tendons from some, mostly prepubertal, mutant animals. In contrast, α1(I)3 collagen from the oim mouse—the only other non-lethal murine OI model studied by similar techniques—has altered stability, fibrillogenesis, collagen–collagen interactions and produces a more consistent and more pronounced disruption of tendon crystallinity. Nevertheless, while the G349C substitution causes moderate or lethal OI, heterozygous oim mice are much less affected. Overall, our results suggest that OI symptoms and phenotype variation in G349C animals are related to abnormal interactions of mutant collagen helices with other matrix molecules or abnormal function of osteoblasts rather than to abnormal structure, physical properties or interactions between mutant collagen helices.

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.

Studies of the local conformational properties of the cell-adhesion domain of collagen type IV in synthetic heterotrimeric peptides.

Collagen type IV is a specialized form of collagen that is found only in basement membranes. It is involved in integrin-mediated cell-adhesion processes, and the responsible binding sites for the alpha1beta1 integrin cell receptor have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. In the most plausible stagger of native collagen type IV the alpha2 chain is the tailing one. This has recently been confirmed by the differentiated binding affinities of synthetic heterotrimeric collagen peptides in which the chains were staggered in this native register as well as in the less plausible alpha1alpha2alpha1' register with an artificial cystine knot. In the present work, two heterotrimeric collagen peptides with chain registers identical to the previous ones were synthesized for fluorescence resonance energy transfer and emission anisotropy measurements, exploiting the native Phe464 in the alpha2 chain as donor and an Ile467Tyr mutation in the alpha1' chain as acceptor fluorophore. This fluorophore pair allowed extraction of more detailed information on the conformational properties of the cell-adhesion epitope incorporated into the central part of the trimeric collagen model peptides. A comparison of the experimentally derived values of the interfluorophore distance and of the orientation factor kappa(2) with the values extracted from the molecular model of the trimer in the native stagger confirmed a triple-helical structure of the adhesion-site portion at low temperature. The thermal unfolding of this central domain was specifically monitored by emission anisotropy, allowing unambiguous assignment of the three structural domains of the trimeric collagen molecules detected by microcalorimetry, with the integrin binding site as the portion of weakest triple-helical stability flanked by two more stable triple-helical regions. The results are consistent with the picture of a conformational microheterogeneity as the responsible property for selective recognition of collagens by interacting proteins.

Identification and characterization of a novel 38.5-kilodalton cell surface protein of Staphylococcus aureus with extended-spectrum binding activity for extracellular matrix and plasma proteins.

The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of S. carnosus. rEmp expressed in Escherichia coli interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a K(d) of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted S. aureus molecules that interact with an extended spectrum of host ligands and thereby contribute to S. aureus pathogenicity.

The 1.8-Å Crystal Structure of a Matrix Metalloproteinase 8-Barbiturate Inhibitor Complex Reveals a Previously Unobserved Mechanism for Collagenase Substrate Recognition

The individual zinc endoproteinases of the tissue degrading matrix metalloproteinase (MMP) family share a common catalytic architecture but are differentiated with respect to substrate specificity, localization, and activation. Variation in domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four distinct classes (Nagase, H., and Woessner, J. F. J. (1999) J. Biol. Chem. 274, 21491-21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-A crystal structure of human neutrophil collagenase (MMP-8) in complex with an active site-directed inhibitor (RO200-1770), we identify and describe new structural determinants for substrate and inhibitor recognition in addition to the primary substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Ala206-Asn218). In stromelysin (MMP-3), competing stabilizing interactions at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of the inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T., Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995) Eur. J. Biochem. 228, 830-841) for binding to MMP-8. The two additional phenyl and piperidyl ring substituents of the inhibitor bind into the S1' and S2' pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond between the O(gamma) group of Ser209 and N(delta)1 of His207 of a symmetry related molecule; this interaction suggests a model for recognition of hydroxyprolines present in physiological substrates. We also identify a collagenase-characteristic cis-peptide bond, Asn188-Tyr189, on a loop essential for collagenolytic activity. The sequence conservation pattern at this position marks this cis-peptide bond as a determinant for triple-helical collagen recognition and processing.

Structural Basis of Collagen Recognition by Integrin α2β1

We have determined the crystal structure of a complex between the I domain of integrin α2β1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin–ligand recognition.

Integrin α2I Domain Recognizes Type I and Type IV Collagens by Different Mechanisms

The collagens are recognized by the alphaI domains of the collagen receptor integrins. A common structural feature in the collagen-binding alphaI domains is the presence of an extra helix, named helix alphaC. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alphaC in the alpha(2)I domain and tested the function of the resultant recombinant protein (DeltaalphaCalpha(2)I) by using a real-time biosensor. The DeltaalphaCalpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alphaC in type I collagen binding and that the collagen binding surface in alpha(2)I domain is located near the metal ion-dependent adhesion site. Previous studies have suggested that the charged amino acid residues, surrounding the metal ion-dependent adhesion site but not interacting with Mg(2+), may play an important role in the recognition of type I collagen. Direct evidence indicating the participation of these residues in collagen recognition has been missing. To test this idea, we produced a set of recombinant alpha(2)I domains with mutations, namely D219A, D219N, D219R, E256Q, D259N, D292N, and E299Q. Mutations in amino acids Asp(219), Asp(259), Asp(292), and Glu(299) resulted in weakened affinity for type I collagen. When alpha(2) D219N and D292N mutations were introduced separately into alpha(2)beta(1) integrin expressed on Chinese hamster ovary cells, no alterations in the cell spreading on type I collagen were detected. However, Chinese hamster ovary cells expressing double mutated alpha(2) D219N/D292N integrin showed remarkably slower spreading on type I collagen, while spreading on type IV collagen was not affected. The data indicate that alpha(2)I domain binds to type I collagen with a different mechanism than to type IV collagen.

Mapping the Collagen-binding Site in the I Domain of the Glycoprotein Ia/IIa (Integrin α2β1)

The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.

Conformational Studies on the Specific Cleavage Site of Type I Collagen (α-1) Fragment (157–192) by Cathepsins K and L by Proton NMR Spectroscopy

Cathepsins K and L are cysteine proteinases which are considered to play an important role in bone resorption. Type I collagen is the most abundant component of the extracellular matrix of bone and regarded as an endogenous substrate for the cysteine proteinases in osteoclastic bone resorption. We have synthesized a fragment of Type I collagen (α-1) (157–192) as a substrate for the cathepsins and found that cathepsins K and L cleave the fragment at different specific sites. The major cleavage sites for cathepsin K were Met159-Gly160, Ser162-Gly163 and Arg165-Gly166, while those for cathepsin L were Gly166-Leu167 and Gln180-Gly181. The structure of the fragment was analyzed in aqueous solution by circular dichroism and proton NMR spectroscopy and the difference in the molecular recognition of collagen by cathepsins K and L was discussed from the structural aspect. ©

Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides.

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.

The von Willebrand Factor A3 Domain Does Not Contain a Metal Ion-dependent Adhesion Site Motif

von Willebrand factor (vWF) is a multimeric plasma protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury. The A3 domain of vWF (vWF-A3) forms the principal binding site for collagens type I and III. We report here the crystal structure of the vWF-A3 domain at 2.2-A resolution. As expected, the structure is similar to the integrin I domain but with several novel features. Sequence alignments had suggested that the domain contained an integrin metal ion-dependent adhesion site (MIDAS) motif, but the crystal structure shows that the motif is modified and that no metal ion is bound. We have introduced mutations into the vestigial MIDAS motif and report that, unlike the I domain of integrin alpha2beta1, vWF-A3 continues to bind collagen after disruption of the motif. We conclude that collagen recognition by vWF-A3 occurs by a mechanism different from that of the integrin alpha2beta1.

X-Ray Crystal Structure of HLA-DR4 (DRA*0101, DRB1*0401) Complexed with a Peptide from Human Collagen II

Genetic predisposition to rheumatoid arthritis (RA) is linked to the MHC class II allele HLA-DR4. The charge of the amino acid at DRβ71 in the peptide-binding site appears to be critical in discriminating DR molecules linked to increased disease susceptibility. We have determined the 2.5 Å x-ray structure of the DR4 molecule with the strongest linkage to RA (DRB1*0401) complexed with a human collagen II peptide. Details of a predicted salt bridge between lysine DRβ71 and aspartic acid at the P4 peptide position suggest how it may participate in both antigen binding and TCR activation. A model is proposed for the DR4 recognition of collagen II (261–273), an antigen immunodominant in human-transgenic mouse models of RA.

Crystal Structure of an Ecotin−Collagenase Complex Suggests a Model for Recognition and Cleavage of the Collagen Triple Helix

The crystal structure of fiddler crab collagenase complexed with the dimeric serine protease inhibitor ecotin at 2.5 A resolution reveals an extended cleft providing binding sites for at least 11 contiguous substrate residues. Comparison of the positions of nine intermolecular main chain hydrogen bonding interactions in the cleft, with the known sequences at the cleavage site of type I collagen, suggests that the protease binding loop of ecotin adopts a conformation mimicking that of the cleaved strand of collagen. A well-defined groove extending across the binding surface of the enzyme readily accommodates the two other polypeptide chains of the triple-helical substrate. These observations permit construction of a detailed molecular model for collagen recognition and cleavage by this invertebrate serine protease. Ecotin undergoes a pronounced internal structural rearrangement which permits binding in the observed conformation. The capacity for such rearrangement appears to be a key determinant of its ability to inhibit a wide range of serine proteases.

Altered cell-matrix contact: a prerequisite for breast cancer metastasis?

The integrins are receptors that regulate interaction between epithelial cells and the extracellular matrix. Previous studies have shown that a reduction in the expression of the alpha2beta1, alpha3beta1, alpha6beta1, alpha(v)beta1 and alpha(v)beta5 integrins in primary breast cancer is associated with positive nodal status. In order to assess the functional significance of altered integrin expression, primary breast cancer cells were derived from individual patients with known tumour characteristics using immunomagnetic separation. Purified human fibronectin, vitronectin, laminin and type IV collagen were used to represent the principal extracellular matrix proteins in an in vitro adhesion assay. Primary breast cancer cells from lymph node-positive patients were significantly less adhesive to each of the matrix proteins studied (P<0.001, Mann-Whitney U-test). Matrix adhesion of primary breast cancer cells from node-negative patients was inhibited by appropriate integrin monoclonal antibodies (P<0.001, paired Wilcoxon test). Adhesion to fibronectin, vitronectin and laminin, but not type IV collagen, was influenced by the inhibitor arginine-glycine-aspartate, suggesting that breast cancer cell recognition of collagen IV is mediated through alternative epitopes. Weak matrix adhesion correlated with loss of integrin expression in tissue sections from corresponding patients assessed using immunohistochemistry. This study demonstrates a link between altered integrin expression and function in primary breast cancers predisposed to metastasize.

β 1 integrins and osteoclast function: Involvement in collagen recognition and bone resorption

The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen binding properties of osteoclast integrins ( α v, α 2, β 1, and β 3). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via α 2β 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved α vβ 3. In receptor-binding assays, the involvement of human recombinant α vβ 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II–V collagen with characteristics similar to those on native monomeric type I collagen. ] Osteoclastic bone resorption in vitro was inhibited (>40%) in the presence of α2 and β 1 antibodies. Using scanning laser confocal microscopy, α vβ 3, α 2, and β 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demontrate that α 2β 1, in addition to α vβ 3, has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.

Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

Specific and charge interactions mediate collagen recognition by oral lactobacilli.

The mechanisms by which oral lactobacilli, one of the three major genera of cariogenic bacteria, attach to tooth surfaces are unknown. We hypothesize that recognition of collagen, the major component of dentin, may be a mechanism which localizes these bacteria to exposed root surfaces as well as to carious lesions which have penetrated the dentin. We found that the majority of oral Lactobacillus spp. strains recognize and bind collagen type I. Binding of 125I-labeled collagen type I to two strains of L. casei rhamnosus has been characterized in some detail. These strains were previously characterized with respect to their attachment to dentin (Switalski and Butcher, 1994). The process of 125I-collagen binding was mediated via specific as well as charge interactions. The putative adhesin-mediated (specific) interaction involved a limited number of bacterial surface components (2 x 10(3)/cell). Under conditions conducive for non-specific interactions (low ionic strength), the binding was higher by an order of magnitude. Collagen binding strains were found to adhere to collagen-coated surfaces, while strains unable to bind collagen adhered to a much lesser extent. Adherence of bacteria to collagen-coated surfaces could be competitively inhibited with collagen. These interactions may target collagen-binding strains of lactobacilli to dentin collagen in the oral cavity and thus play a role in the pathogenesis of root surface and/or coronal caries. Interference with this collagen-mediated attachment of lactobacilli may provide effective means of caries control, particularly in view of the fact that other oral acidogenic microbiota also interact with collagen.

Importance of triple helical conformation in the recognition of collagen V binding sites by integrins

Importance of triple helical conformation in the recognition of collagen V binding sites by integrins

Autoimmune recognition of cartilage collagens.

Recent advances in basic research on the immune system and molecular biology of cartilage components have greatly increased our understanding of the role of autoimmunity in inflammatory diseases affecting joints, particularly rheumatoid arthritis. Many of these diseases are common and their complex pathogenesis probably involves a large number of genes polymorphic in the population as well as environmental factors. Characteristic features of inflammatory arthritis include expansion of the synovial tissue into a pannus containing lymphocytes and macrophages, autoimmune reactions against cartilage antigens, and erosion of cartilage. Since hyaline cartilage of the articular surfaces is the only structure within the joint known to contain joint-specific antigens this tissue is the prime suspect as the target of the autoimmune This review will first present the capacity of the immune system to discriminate between self and non-self structures, and then summarize our current understanding of the structures of cartilage collagens. Subsequently we will discuss how the immune system normally interacts with cartilage and how such interactions can lead to arthritis. We propose that collagen-induced arthritis (CIA) is valuable for understanding the autoimmune recognition of cartilage collagen which precedes the outbreak of arthritis and may perpetuate its chronicity, and serves as an animal model of rheumatoid arthritis.

Identification of an immunodominant type‐II collagen peptide recognized by T cells in H‐2q mice: self tolerance at the level of determinant selection

The T cell recognition of type-II collagen (CII) in H-2q mice, susceptible to CII-induced arthritis, was analyzed. With the use of T cell hybridomas derived from rat CII-immunized mice, a peptide corresponding to amino acids 245-270 on chick CII was found to harbor a T cell epitope which is present on heterologous CII (chick, rat, human, and bovine CII) but not on autologous CII. It was shown that this epitope was located within amino acids 260-270, although flanking regions in either direction were necessary for proper recognition. A peptide corresponding to human CII (256-270) was used for further studies. A single amino acid difference at position 266 between mouse CII (aspartic acid) and heterologous CII (glutamic acid) strongly influenced recognition of this peptide. No response towards the mouse peptide was seen with any of the T cell hybridomas. Inhibition studies revealed that the mouse peptide did not bind as well to major histocompatibility complex as the corresponding heterologous peptide. Both peptides gave rise to a T cell response after immunization. However, immunization with the heterologous peptide resulted in a response strictly directed to rat CII and the immunogen while immunization with the autologous peptide elicited T cells which reacted in a heteroclitic fashion, with a stronger response to the heterologous peptide than to the autologous peptide, and did respond to rat CII but not to mouse CII. We suggest that aspartic acid in position 266 results in a cryptic determinant in mouse CII which is neither recognized after CII immunization nor capable of tolerance induction. A glutamic acid at position 266, however, gives rise to an immunodominant epitope which is recognized by a large proportion of the T cells activated after immunization with heterologous CII.