Type VI microfilaments interact with a specific region of banded collagen fibrils in skin.

Immunolocalization studies demonstrate that Type VI collagen forms a flexible network that interweaves among collagen fibrils in the dermis of skin as well as in other loose connective tissues. Although binding of Type VI collagen with other matrix components has been suggested, no structural evidence supporting these studies has been reported. In this study, we demonstrate that Type VI microfilaments consistently crossbanded collagen fibrils near the "d" band, indicating that the interaction of Type VI collagen with banded fibrils is not passive. This "d" band is also the location of the binding domain of decorin to banded fibrils, suggesting that decorin mediates the interaction of Type VI microfilaments with banded fibers. Examination of the architecture of the Type VI network in a decorin nullizygous mouse demonstrates a continuance of this specific interaction, indicating that the association is not entirely dependent on the presence of decorin. At least one other component, whose identity is uncertain, persists near the "d" band, which may also serve to mediate the attachment of Type VI collagen to collagen fibrils.

Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites.

Secondary structure and shape of plasma sex steroid-binding protein--comparison with domain G of laminin results in a structural model of plasma sex steroid-binding protein.

We have analyzed the secondary structure, shape and dimensions of plasma sex steroid-binding protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of steroid-binding activity. As the SBP sequence shows similarity with domains of several multidomain proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the steroid-binding site located in the center of the rod-like particle.

The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.

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 cell-specific 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.

Triple helix formation of procollagen type I can occur at the rough endoplasmic reticulum membrane.

One key problem in understanding the biosynthesis of collagens remains the assembly of the three alpha-chains. How and when are the different gene products selected, aligned, and folded into a triple helix? As the spatial arrangement during biosynthesis might be important, we concentrated on whether the rough endoplasmic reticular membrane is involved in this process. Microsomes were prepared from biosynthetically labeled chick tendon fibroblasts. Vesicles were spread as a monomolecular film which was then transferred over several compartments of a filmbalance containing fresh subphase. Fluorograms of the surface film showed that the monolayer contains procollagen chains. When the monolayer was transferred onto a chymotrypsin/trypsin-containing subphase, the gel bands of the proalpha-chains were shifted into the position of mature alpha-chains, indicating that only the propeptides were digested and the collagenous regions were protected due to triple helix formation. Our results suggest that newly synthesized proalpha-chains can associate as trimers and fold into a triple helical conformation while they are still associated with the membranes of the rough endoplasmic reticulum. These processes also occur when interchain disulfide linkage is inhibited, indicating that chain selection and registration is not dependent on formation of covalent bonds among the carboxyl propeptides.