Fibrillin is a large (relative molecular mass 350,000) glycoprotein which can be isolated from fibroblast cell cultures and is a component of the microfibrils that are ubiquitous in the connective tissue space. The microfibrils of the suspensory ligament of the lens as well as the elastic fibre microfibrils of the blood vessel wall are composed of fibrillin. The ocular and cardiovascular manifestations of the Marfan syndrome are consistent with a defect in the gene coding for a structural constituent of these connective tissues. Immunohistological experiments have recently implicated fibrillin microfibrils in the pathogenesis of the Marfan syndrome. Genetic linkage data localizing the Marfan gene to chromosome 15 and the in situ hybridization of fibrillin complementary DNA to 15q21.1 together support fibrillin as a candidate Marfan gene. As a first step towards investigating the function of fibrillin in the architecture and development of connective tissues and its relationship to the Marfan syndrome, we report the cloning and partial sequencing of fibrillin cDNA.

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.

We have identified a novel missense mutation in a pseudoachondroplasia (PSACH) patient in one of the type III repeats of cartilage oligomeric matrix protein (COMP). Enlarged lamellar rough endoplasmic reticulum vesicles were shown to contain accumulated COMP along with type IX collagen, a cartilage-specific component. COMP was secreted and assembled normally into the extracellular matrix of tendon, demonstrating that the accumulation of COMP in chondrocytes was a cellspecific phenomenon. We believe that the intracellular storage of COMP causes a nonspecific aggregation of cartilage-specific molecules and results in a cartilage matrix deficient in required structural components leading to impaired cartilage growth and maintenance. These data support a common pathogenetic mechanism behind two clinically related chondrodysplasias, PSACH and multiple epiphyseal dysplasia. Mutations in COMP,1 cartilage oligomeric matrix protein, have been associated with a bone dysplasia family that includes pseudoachondrodysplasia (PSACH) and multiple epiphyseal dysplasia (MED). PSACH and MED (EDM1) were localized to chromosome 19p13.1, the region that contains the gene for COMP (1-4), and specific base substitutions, deletions, and duplications have been subsequently identified (5, 6). Both PSACH and MED are inherited as dominant disorders.The clinical features of the two diseases are similar and can range from mild to severe forms (7). PSACH is characterized by a disproportionate short stature and joint laxity with a waddling gait that appears with the onset of walking. MED patients generally present with mild short stature and hip pain later in childhood. The radiological abnormalities of MED are restricted to the epiphyses whereas there is additional involvement of metaphyses and the spinal column in patients with PSACH. Patients with both disorders manifest symptoms of precocious osteoarthritis. The diseases appear to be allelic variants resulting from different mutations in the same gene.Another extracellular matrix component of cartilage, type IX collagen, has also been implicated in MED. This form of MED (EDM2) affects primarily the growth centers of the knees and has been mapped to chromosome 1p32 (8) in a family that had previously shown no linkage to COMP (chromosome 19p13). COL9A2 was also mapped to this locus (9). The major complaint of this MED family was early onset osteoarthritis, especially in the knees. Only a few individuals exhibited mild short stature, distinguishing this family from others with MED. Recently, a mutation in the ␣2 chain of type IX collagen was identified in this large family (10). It is not clear how mutations in these two genes result in a similar phenotype.COMP is the fifth member of the thrombospondin (TSP) family of extracellular matrix glycoproteins (3). It was originally discovered in cartilage extracts and has been immunolocalized to developing as well as mature cartilage (11,12). COMP has also been found within and around tendon bundles (13). Rotary shadow imaging shows the molecule to b...

Cartilage oligomeric matrix protein (COMP) is a member of the thrombospondin family of extracellular matrix glycoproteins. All members of the family contain a highly conserved region of thrombospondin type 3 sequence repeats that bind calcium. A mutation in COMP previously identified in a patient with pseudoachondroplasia resulted in abnormal sequestration of COMP in distinctive rER vesicles. The mutation, Asp-446 3 Asn, is located in the type 3 repeats of the molecule. This region was expressed in a mammalian culture with and without the mutation to study the structural or functional properties associated with the mutation. The biophysical parameters of the mutant peptide were compared with those of the wild type and revealed the following difference: secondary structural analysis by circular dichroism showed more ␣-helix content in the wild-type peptides. The calcium binding properties of the two peptides were significantly different; there were 17 calcium ions bound/wild-type COMP3 peptide compared with 8/mutant peptide. In addition, wild-type COMP3 had a higher affinity for calcium and bound calcium more cooperatively. Calcium bound by the wild-type peptide was reflected in a structural change as indicted by velocity sedimentation. Thus, the effect of the COMP mutation appears to profoundly alter the calcium binding properties and may account for the difference observed in the structure of the type 3 domain. Furthermore, the highly cooperative binding of calcium to COMP3 suggests that these type 3 sequence repeats form a single protein domain, the thrombospondin type 3 domain.