Birgit Kobbe

Collagen XXVIII, a Novel von Willebrand Factor A Domain-containing Protein with Many Imperfections in the Collagenous Domain

Here we describe a novel collagen belonging to the class of von Willebrand factor A (VWA) domain-containing proteins. This novel protein was identified by screening the EST data base and was subsequently recombinantly expressed and characterized as an authentic tissue component. The COL28A1 gene on human chromosome 7p21.3 and on mouse chromosome 6A1 encodes a novel protein that structurally resembles the beaded filament-forming collagens. The collagenous domain contains several very short interruptions arranged in a repeat pattern. As shown for other novel minor collagens, the expression of collagen XXVIII protein in mouse is very restricted. In addition to small amounts in skin and calvaria, the major signals were in dorsal root ganglia and peripheral nerves. By immunoelectron microscopy, collagen XXVIII was detected in the sciatic nerve, at the basement membrane of certain Schwann cells surrounding the nerve fibers. Even though the protein is present in the adult sciatic nerve, collagen XXVIII mRNA was only detected in sciatic nerve of newborn mice, indicating that the protein persists for an extended period after synthesis.

Three Novel Collagen VI Chains with High Homology to the α3 Chain

Here we describe three novel collagen VI chains, α4, α5, and α6. The corresponding genes are arranged in tandem on mouse chromosome 9. The new chains structurally resemble the collagen VI α3 chain. Each chain consists of seven von Willebrand factor A domains followed by a collagenous domain, two C-terminal von Willebrand factor A domains, and a unique domain. In addition, the collagen VI α4 chain carries a Kunitz domain at the C terminus, whereas the collagen VI α5 chain contains an additional von Willebrand factor A domain and a unique domain. The size of the collagenous domains and the position of the structurally important cysteine residues within these domains are identical between the collagen VI α3, α4, α5, and α6 chains. In mouse, the new chains are found in or close to basement membranes. Collagen VI α1 chain-deficient mice lack expression of the new collagen VI chains implicating that the new chains may substitute for the α3 chain, probably forming α1α2α4, α1α2α5, or α1α2α6 heterotrimers. Due to a large scale pericentric inversion, the human COL6A4 gene on chromosome 3 was broken into two pieces and became a non-processed pseudogene. Recently COL6A5 was linked to atopic dermatitis and designated COL29A1. The identification of novel collagen VI chains carries implications for the etiology of atopic dermatitis as well as Bethlem myopathy and Ullrich congenital muscular dystrophy.

Molecular Structure and Tissue Distribution of Matrilin-3, a Filament-forming Extracellular Matrix Protein Expressed during Skeletal Development

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 SDS-polyacrylamide 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 – adaptor proteins in the extracellular matrix

The matrilins form a four‐member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen‐containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin‐3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin‐3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.

Primary structure of matrilin-3, a new member of a family of extracellular matrix proteins related to cartilage matrix protein (matrilin-1) and von Willebrand factor

A mouse cDNA encoding for matrilin‐3, the third member of the novel matrilin family of extracellular matrix proteins, was cloned. The protein precursor of 481 amino acids consists of a putative signal peptide, a short positively charged sequence, a single vWFA‐like domain followed by four epidermal growth factor‐like modules and a potential coiled‐coil α‐helical oligomerization domain at the C‐terminus. It is the smallest member of the matrilin family with a predicted M r of the mature protein of 48 902. The primary structure of a C‐terminal portion of 310 amino acids of the human matrilin‐3 was determined and showed a sequence identity to the mouse matrilin‐3 of 84.8%. Northern blot hybridization of mouse matrilin‐3 mRNA showed a 2.9 kb mRNA expressed in sternum, femur and trachea and indicates a cartilage‐specific expression.

Matrilin-3 Is Dispensable for Mouse Skeletal Growth and Development

ABSTRACT Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.

MATRILIN-4, A NEW MEMBER OF THE MATRILIN FAMILY OF EXTRACELLULAR MATRIX PROTEINS

Mouse cDNA encoding for matrilin‐4 was cloned and the primary structure of this fourth member of the matrilin family was deduced from the nucleotide sequence. The protein precursor of 624 amino acids consists of a putative signal peptide, two vWFA‐like domains linked by four epidermal growth factor‐like modules and a potential coiled‐coil α‐helical oligomerization domain at the C‐terminus. The predicted M r of the mature protein is 66 442. Expression in lung, brain, sternum, kidney and heart was detected by Northern blot analysis of mouse mRNA. Additionally an alternatively spliced mRNA lacking the sequence coding for the first vWFA domain was found in 7 weeks old mice leading to a protein precursor of 434 amino acids and a predicted M r of the mature protein of 45 468.

The knee osteoarthritis susceptibility locus DVWA on chromosome 3p24.3 is the 5′ part of the split COL6A4 gene

In a recent study the DVWA gene located on human chromosome 3p24.3 was identified as a susceptibility locus for knee osteoarthritis in Japanese and Chinese patients (Miyamoto, Y., Shi, D., Nakajima, M., Ozaki, K., Sudo, A., Kotani, A., Uchida, A., Tanaka, T., Fukui, N., Tsunoda, T., Takahashi, A., Nakamura, Y., Jiang, Q., Ikegawa, S., 2008. Common variants in DVWA on chromosome 3p24.3 are associated with susceptibility to knee osteoarthritis. Nat. Genet. 40, 994-998). The authors concluded that DVWA codes for a novel protein containing two von Willebrand factor A (VWA) domains without a signal peptide sequence. The experimental data provided in this interesting study led to the suggestion of a mechanism for the etiology of the disease, based on an interaction between DVWA protein and beta-tubulin. More recently, no significant association between DVWA and osteoarthritis was found in UK patient samples (Valdes, A.M., Spector, T.D., Doherty, S., Wheeler, M., Hart, D.J., Doherty, M., 2008. Association of the DVWA and GDF5 polymorphisms with osteoarthritis in UK populations. Ann. Rheum. Dis. Dec 3. [Epub ahead of print]), but a meta-analyses with data from individuals of white European descent from the Netherlands, the UK, Spain and Greece and the original Japanese and Chinese cohort provided evidence for a global association of one of the polymorphisms, a cysteine to tyrosine exchange (rs7639618) (Meulenbelt, I., Chapman, K., Dieguez-Gonzalez, R., Shi, D., Tsezou, A., Dai, J., Malizos, K.N., Kloppenburg, M., Carr, A., Nakajima, M., van der Breggen, R., Lakenberg, N., Gomez-Reino, J.J., Jiang, Q., Ikegawa, S., Gonzalez, A., Loughlin, J., Slagboom, E.P., 2009. Large replication study and meta-analyses of DVWA as an osteoarthritis susceptibility locus in European and Asian populations. Hum. Mol. Genet. 8, 1518-1523). However, there was no independent association with knee osteoarthritis in Europeans. Here we present information that the newly identified DVWA represents the human gene coding for the collagen VI alpha 4 chain, which could point to a more complex disease mechanism.

Identification and characterization of AMACO, a new member of the von Willebrand factor A-like domain protein superfamily with a regulated expression in the kidney

The genes coding for human and mouse AMACO, an extracellular matrix protein containing VWA-like domains related to those in MAtrilins and COllagens, were detected in databases, the cDNAs were cloned, and the primary structures were deduced from the nucleotide sequences. The genes consist of 14 exons and have a similar exon/intron organization. The protein consists of a signal peptide sequence, an N-terminal VWA domain connected to two additional, tandem VWA domains by a cysteine-rich sequence and an epidermal growth factor (EGF)-like domain. The C terminus is made up of another EGF-like domain followed by a unique sequence present in mouse, but absent in human. The predicted molecular weight of the proteins is 79,485 in human and 83,024 in mouse. Full-length AMACO was expressed in 293-EBNA cells, purified by use of an affinity tag and subjected to biochemical characterization. Both monomers and aggregates of AMACO were recovered, as shown by electron microscopy and SDS-PAGE. AMACO was found in the media of a variety of established cell lines of both fibroblast and epithelial origin. In the matrix formed by 293-EBNA cells overexpressing the protein, AMACO was deposited in patchy structures that were often cell-associated. Affinity-purified antibodies detect expression in cartilage and expression associated with certain basement membranes. In the kidney of adult mice, a second promoter located in intron 4 is active. If the resulting transcript is translated it could not yield a secreted protein because of the lack of a signal peptide sequence. The developmental switch from an AMACO mRNA, expressed by the newborn kidney, to the truncated transcript found in the adult kidney indicates an unusual regulation of AMACO expression.

Zebrafish (Danio rerio) matrilins: shared and divergent characteristics with their mammalian counterparts

We have cloned the cDNAs of the zebrafish (Danio rerio) members of the matrilin family of extracellular adaptor proteins. In contrast to mammals, no orthologue of matrilin-2 was found in zebrafish, either by RT (reverse-transcriptase) PCR using degenerated primers or by screening the databases (Ensembl and NCBI); however, two forms of matrilin-3, matrilin-3a and -3b, were present. The identity with the mammalian matrilins is from more than 70% for the VWA (von Willebrand factor A)-like domains to only 28% for the coiled-coil domains of matrilin-3a and -3b. In all zebrafish matrilins we found a greater variety of splice variants than in mammals, with splicing mainly affecting the number of EGF (epidermal growth factor)-like repeats. The exon-intron organization is nearly identical with that of mammals, and also the characteristic AT-AC intron interrupting the exons coding for the coiled-coil domain is conserved. In the matrilin-3b gene a unique exon codes for a proline- and serine/threonine-rich domain, possibly having mucin-like properties. The matrilin-1 and -3a genes were mapped to chromosome 19 and 20 respectively by the radiation hybrid method. The temporal and spatial expression of zebrafish matrilins is similar to that seen in the mouse. Zebrafish matrilin-4 is highly expressed as early as 24 hpf (h post fertilization), whereas the other matrilins show peak expression at 72 hpf. By immunostaining of whole mounts and sections, we found that matrilin-1 and -3a show predominantly skeletal staining, whereas matrilin-4 is more widespread, with the protein also being present in loose connective tissues and epithelia.