ABSTRACT
The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.
Subject(s)
Cartilage/metabolism , Extracellular Matrix Proteins/biosynthesis , Extremities/embryology , Glycoproteins/biosynthesis , Heart/embryology , Animals , Bone and Bones/embryology , Bone and Bones/metabolism , Embryonic and Fetal Development , Matrilin Proteins , Mice , Myocardium/metabolismABSTRACT
Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.
Subject(s)
Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , Animals , Base Sequence , Connective Tissue/metabolism , DNA Primers , Extracellular Matrix Proteins/genetics , Glycoproteins/genetics , Matrilin Proteins , Mice , Microscopy, Fluorescence , Protein Processing, Post-Translational , RNA, Messenger/geneticsABSTRACT
A mouse cDNA encoding a novel member of the von Willebrand factor type A-like module superfamily was cloned. The protein precursor of 956 amino acids consists of a putative signal peptide, two von Willebrand factor type A-like domains connected by 10 epidermal growth factor-like modules, a potential oligomerization domain, and a unique segment, and it contains potential N-glycosylation sites. A sequence similarity search indicated the closest relation to the trimeric cartilage matrix protein (CMP). Since they constitute a novel protein family, we introduce the term matrilin-2 for the new protein, reserving matrilin-1 as an alternative name for CMP. A 3. 9-kilobase matrilin-2 mRNA was detected in a variety of mouse organs, including calvaria, uterus, heart, and brain, as well as fibroblast and osteoblast cell lines. Expressed human and rat cDNA sequence tags indicate a high degree of interspecies conservation. A group of 120-150-kDa bands was, after reduction, recognized specifically with an antiserum against the matrilin-2-glutathione S-transferase fusion protein in media of the matrilin-2-expressing cell lines. Assuming glycosylation, this agrees well with the predicted minimum Mr of the mature protein (104,300). Immunolocalization of matrilin-2 in developing skeletal elements showed reactivity in the perichondrium and the osteoblast layer of trabecular bone. CMP binds both collagen fibrils and aggrecan, and because of the similar structure and complementary expression pattern, matrilin-2 is likely to perform similar functions in the extracellular matrix assembly of other tissues.
