Mutations in type 3 repeats of cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We expressed recombinant wildtype COMP that showed structural and functional properties identical to COMP isolated from cartilage. A fragment encompassing the eight type 3 repeats binds 14 calcium ions with moderate affinity and high cooperativity and presumably forms one large disulfidebonded folding unit. A recombinant PSACH mutant COMP in which Asp-469 was deleted (D469⌬) and a MED mutant COMP in which Asp-361 was substituted by Tyr (D361Y) were both secreted into the cell culture medium of human cells. Circular dichroism spectroscopy revealed only small changes in the secondary structures of D469⌬ and D361Y, demonstrating that the mutations do not dramatically affect the folding and stability of COMP. However, the local conformations of the type 3 repeats were disturbed, and the number of bound calcium ions was reduced to 10 and 8, respectively. In addition to collagen I and II, collagen IX also binds to COMP with high affinity. The PSACH and MED mutations reduce the binding to collagens I, II, and IX and result in an altered zinc dependence. These interactions may contribute to the development of the patient phenotypes and may explain why MED can also be caused by mutations in collagen IX genes.

The complement system represents an evolutionary old and significant part of the innate immune system involved in protection against invading microorganisms. Here, we show that the anaphylatoxin C3a and its inactivated derivative C3a-desArg are antibacterial, demonstrating a previously unknown direct antimicrobial effect of complement activation. The C3a peptide, as well as functional epitopes in the sequence, efficiently killed the Gramnegative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Enterococcus faecalis. In mice, a C3a-derived peptide suppressed infection by Gram-positive Streptococcus pyogenes bacteria. Fluorescence and electron microscopy demonstrated that C3a binds to and induces breaks in bacterial membranes. C3a was also found to induce membrane leakage of liposomes. These findings provide an interesting link between the complement system and antimicrobial peptides, which are two important branches of innate immunity.bacteria ͉ inflammation ͉ innate immunity

Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDSpolyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.The matrilins constitute a recently discovered family of noncollagenous proteins (1) belonging to the von Willebrand factor A (vWFA) 1 -like domain superfamily. To date, there are four matrilins known. Matrilin-2 (2, 3) and matrilin-4 (4, 5) have a broad tissue distribution, whereas the expression of matrilin-1 (also known as cartilage matrix protein) (6 -8) and matrilin-3 (9 -11) is more restricted to skeletal tissues. The division of the family into two subgroups can also be concluded from evolutionary studies (1). The descent from a common ancestor and the divergence through duplication of whole domains indicates the possibility of the different family members providing similar functions in different tissues. The at least partially coordinated expression of matrilin-1 and -3 gains further functional significance through the recent discovery of hetero-oligomers formed by matrilin-1 and -3 in epiphyseal cartilage of fetal calf femur (12).Matrilin-3 has most features of the modular structure typical for matrilins and consists of an N-terminal vWFA-like domain, four EGF-like domains, and a C-terminal ␣-helical coiled-coil oligomerization domain (9 -11), but it lacks the second vWFAlike domain that is present in all the other matrilins. Similarly, a unique mouse matrilin-4 splice variant lacking the N-terminal vWFA-like domain was recently identified (4). In addition, matrilin-3 possesses a domain with a high content of positively charged amino acids between the N-terminal vWFA-like domain ...

Matrilin-4 is the most recently identified member of the matrilin family of von Willebrand factor A-like domain containing extracellular matrix adapter proteins. Full-length matrilin-4 was expressed in 293-EBNA cells, purified using affinity tags, and subjected to biochemical characterization. The largest oligomeric form of recombinantly expressed full-length matrilin-4 is a trimer as shown by electron microscopy, SDS-polyacrylamide gel electrophoresis, and mass spectrometry. Proteolytically processed matrilin-4 species were also detected. The cleavage occurs in the short linker region between the second von Willebrand factor A-like domain and the coiled-coil domain leading to the release of large fragments and the formation of dimers and monomers of intact subunits still containing a trimeric coiled-coil. In immunoblots of calvaria extracts similar degradation products could be detected, indicating that a related proteolytic processing occurs in vivo. Matrilin-4 was first observed at day 7.5 post-coitum in mouse embryos. Affinity-purified antibodies detect a broad expression in dense and loose connective tissue, bone, cartilage, central and peripheral nervous systems and in association with basement membranes. In the matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells.

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Human bone sialoprotein (BSP) comprises 15% of the total noncollagenous proteins in bone and is thought to be involved in bone mineralization and remodeling. Recent data suggest a role for BSP in breast cancer and the development of bone metastases. We have produced fulllength recombinant BSP in a human cell line and purified the protein from human bone retaining the native structure with proper folding and post-translational modifications. Mass spectrometry of bone-derived BSP revealed an average mass of 49 kDa and for recombinant BSP 57 kDa. The post-translational modifications contribute 30 -40%. Carbohydrate analysis revealed 10 different complex-type N-glycans on both proteins and eight different O-glycans on recombinant BSP, four of those were found on bone-derived BSP. We could identify eight threonines modified by O-glycans, leaving the C terminus of the protein free of glycans. The recombinant protein showed similar secondary structures as bone-derived BSP. BSP was visualized in electron microscopy as a globule linked to a thread-like structure. The affinity for hydroxyapatite was higher for bonederived BSP than for recombinant BSP. Cell adhesion assays showed that the binding of BSP to cells can be reversibly diminished by denaturation. Bone sialoprotein (BSP)1 is a noncollagenous protein that was first isolated from bovine bone (1). The protein is very abundant as it comprises about 15% of the total noncollagenous proteins in bone. After cleavage of the signal peptide sequence the polypeptide chain of human BSP contains 301 amino acids. According to the cDNA sequence the molecular mass of the core protein is 33.6 kDa (2). However, after separation on SDS-PAGE the protein could be identified as an 80-kDa band (3, 4). The reason for these different sizes is most likely the high content of post-translational modifications, where carbohydrates were shown to contribute about 50% to the mass of the protein (5). However, the nature of the carbohydrates on human BSP has not been described. Additionally the protein was shown to be sulfated and phosphorylated (6, 7). Close to the C terminus the molecule contains an RGD cell-binding motif that enables the protein to bind integrin ␣ v ␤ 3 (8). There are three tyrosine-rich regions and two clusters rich in glutamic acids (9).Bone sialoprotein is expressed by most cell types in bone, cartilage, and teeth. In the mineralized bone matrix the highest BSP concentrations were found in areas where bone is newly synthesized or remodeled (10, 11). Outside of the skeleton the protein was found in placental trophoblasts (12) and in platelets (13). Additionally, BSP is expressed in certain carcinomas that predominantly metastasize to bone, i.e. breast, prostate, lung, thyroid tumors, as well as multiple myeloma and neuroblastoma. Bone metastases of breast and prostate cancer patients expressed significantly more BSP than visceral metastases (14).The function of BSP in bone and cartilage is still not fully understood. Using the RGD sequence the protein can bind cell surfaces o...

Group G streptococcus (GGS) is a human pathogen of emerging clinical significance. It causes skin and soft tissue infections, occasionally resulting in life-threatening conditions such as sepsis and necrotizing fasciitis. We recently identified FOG, a novel surface protein of GGS with fibrinogen binding and immune evasion properties. Here we investigated the role of FOG in streptococcal primary adhesion to host tissue. A FOG-expressing clinical isolate adhered more efficiently to human skin biopsies ex vivo and to the murine dermis in vivo than a FOG-deficient strain. Scanning and transmission electron microscopy of skin specimens exhibited that this property was assigned to the ability of FOG to interact with collagen I, a major interstitial component of the dermis. Overlay experiments with human skin extracts and radiolabeled FOG followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis identified both the ␣1-and ␣2-chains of collagen I as targets for FOG. Transmission electron microscopy of the molecular complexes revealed thread-like FOG molecules binding via their NH 2 termini to distinct sites on collagen I monomers and fibrils. The results demonstrate that FOG is important for GGS adhesion in vivo, implying a pathogenic role for this surface protein.Streptococcus dysgalactiae equisimilis, a group G streptococcus (GGS), 5 can cause mild infections like pharyngitis and more severe skin infections such as erysipelas and abscess formation. Deep soft tissue infections may result in life-threatening conditions such as sepsis and necrotizing fasciitis (for references see Ref. 1). Although certain cases of rheumatic fever have been linked to pharyngeal carriage of GGS (2), primary infections of the skin with these pathogens were often found to precede serious invasive conditions and bacteremia (3). Thus, the skin is to be considered an important site of entry. The clinical relevance of skin infections caused by GGS is underlined by epidemiological reports indicating that GGS are at least as frequent as group A streptococci (GAS) in such diseases (4, 5).Primary adhesion to the host tissue is a crucial initial step in bacterial infection. The firm interaction enables the bacteria to persist at the site of infection and subsequently colonize specific locations in the tissue. Certain streptococcal surface components with the potential to adhere to host tissues have been identified previously (for references see Refs. 6 and 7). For GAS a mechanism has been postulated in which lipoteichoic acid acts in concert with bacterial surface proteins to facilitate adhesion to host cells (8 -10). Frequently fibronectin has a key function by bridging bacterial adhesins and eukaryotic integrins (11-15). Interestingly, some of the M proteins, which are important multifunctional virulence factors of GAS, meet the criteria of adhesins (for references see Refs. 6 and 7). As an example, M1 protein involves both fibronectin and laminin in adhesion to human lung epithelial cells (16).A recent study...

We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.