Porcine factor V: cDNA cloning, gene mapping, three-dimensional protein modeling of membrane binding sites and comparative anatomy of domains

Factor V is a plasma protein essential for blood coagulation. This protein is involved in activated protein C resistance, the most common inherited thrombotic disorder known. We utilized the polymerase chain reaction to clone the porcine factor V gene by generating overlapping clones amplified with primers chosen by comparison with known nucleotide sequences. The porcine factor V cDNA contig encodes a predicted 2258-amino acid protein, making it the largest in comparison to the bovine, human, and murine proteins. Porcine factor V has the highest level of homology with bovine factor V, but also has high levels of conservation of important residues with all the species. Radiation hybrid mapping assigned the porcine factor V gene to chromosome 4. Three-dimensional models of factor V were generated and used to analyze membrane-binding sites in terms of conserved, and therefore likely important residues. Received 3 October 2000; revised 23 November 2000; accepted 6 December 2000     

Noncollagenous, nonproteoglycan macromolecules of cartilage

Extracellular matrix comprises approximately 90% of cartilage, with collagens and proteoglycans making up the bulk of the tissue. In recent years, several abundant cartilage proteins that are neither collagens nor proteoglycans have been characterized in detail. The putative roles of these proteins range from involvement in matrix organization or matrix-cell signaling (PRELP, chondroadherin, cartilage oligomeric protein and cartilage matrix protein) through to molecules that are likely to be involved with modulation of the chondrocyte phenotype (CD-RAP, CDMPs, chondromodulin and pleiotrophin). Other molecules, such as the cartilage-derived C-type lectin and cartilage intermediate layer protein have no role as yet. Due to the difficulties associated with experimentally manipulating a tissue that is 90% extracellular matrix in a manner that can be readily transferred to the whole organism, many of these molecules have been focused on by a surprisingly small number of researchers. This review focuses on newly discovered proteins and glycoproteins in cartilage, with a bias towards those that have structural roles or that are unique to cartilage. Received 7 January 1999; accepted 11 March 1999     

Independent modulation of collagen fibrillogenesis by decorin and lumican

The leucine-rich proteoglycans (also known as "small, leucine-rich proteoglycans," or SLRPs) lumican and decorin are thought to be involved in the regulation of collagen fibril assembly. Preparation of these proteoglycans in chemical amounts without exposure to denaturants has recently been achieved by infecting HT-1080 cells with vaccinia virus that contains an expression cassette for these molecules. Addition of lumican and decorin to a collagen fibrillogenesis assay based on turbidity demonstrated that lumican accelerated initial fibril formation while decorin retarded initial fibril formation. At the end of fibrillogenesis, both proteoglycans resulted in an overall reduced turbidity, suggesting that fibril diameter was lower. The presence of both proteoglycans had a synergistic effect, retarding fibril formation to a greater degree than either proteoglycan individually. Competitive binding studies showed that lumican did not compete for decorin-binding sites on collagen fibrils. Both proteoglycans increased the stability of fibrils to thermal denaturation to approximately the same degree. These studies show that lumican does not compete for decorin-binding sites on collagen, that decorin and lumican modulate collagen fibrillogenesis, and that, in the process, they also enhance collagen fibril stability.     

The link proteins

Aggregates of chondroitin-keratan sulfate proteoglycan (aggrecan) and hyaluronic acid (hyaluronan) are the major space-filling components of cartilage. A glycoprotein, link protein (LP; 40–48 kDa) stabilizes the aggregate by binding to both hyaluronic acid and aggrecan. In the absence of LP, aggregates are smaller (as estimated by rotary shadowing of electron micrographs) and less stable (they dissociate at pH 5) than they are in the presence of LP. The proteoglycan aggregate, including LP, is dissociated in the presence of chaotropes such as 4 M guanidine hydrochloride. On removal of the chaotrope, the complex will reassociate. This forms the basis of the isolation of LP from cartilage and has been described in detail elsewhere. Tryptic digestion of the proteoglycan aggregates results in a high molecular weight product that consists of hyaluronic acid to which is bound LP and the N-terminal globular domain of aggrecan (hyaluronic acid binding region; HABR) in a 11 stoichiometry. The amino acid sequences of LP and HABR are surprisingly similar. The amino acid sequence can be divided into three domains; an N-terminal domain that falls into the immunoglobulin super-family and two C-terminal domains that are similar to each other. The DNA structure echoes this similarity, in that the major domains are reflected in three separate exons in both LP and HABR. The two C-terminal domains are largely responsible for the association with HA and are related to two recently described hyaluronate-binding proteins, CD44 and TSG-6. A variety of approaches, including analysis of the forms of LP found in vivo, rotary shadowing and analysis of the sequence in the immunoglobulin-like domain, have shed considerable light on the structure-function relationships of LP. This review describes the structure and function of LP in detail, focusing on what can be inferred from the similarity of LP, HABR and related molecules such as immunoglobulins and lymphocyte HA-receptors.