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 ...