Type VI Collagen Anchors Endothelial Basement Membranes by Interacting with Type IV Collagen

Abstract

Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes. To identify type VI collagen binding proteins, the amino-terminal domains of the ␣1(VI) and ␣2(VI) chains and a part of the carboxyl-terminal domain of the ␣3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library. Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins. The amino-terminal domain of ␣1(VI) was shown to interact with the carboxylterminal globular domain of type IV collagen. The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies. Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy. The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.Type VI collagen filaments are ubiquitous. They are present in all connective tissues that contain type I and type III collagen fibers and in cartilage, a tissue that contains predominantly type II collagen. The major functions that have been suggested for type VI collagen filamentous networks are as a substrate for cell attachment and as an anchoring meshwork that connects collagen fibers, nerves, and blood vessels to the surrounding matrix (1, 2). This implies that not only is there an interaction, either direct or indirect, with the type I/III collagen fibers but also that there is an interaction with components of endothelial basement membranes.Matrix components that have been shown to interact with type VI collagen in vitro include proteoglycans, collagens, hyaluronan, heparin, and integrins.Proteoglycans appear to be particularly important, since cell surface-, basement membrane-, and collagen fibril-associated proteoglycans have all been reported to bind to various forms of type VI collagen. Decorin is a small dermatan sulfate proteoglycan that binds to fibrillar collagens (3). It was shown that the leucine-rich module of the core protein bound to type VI and that the binding could be inhibited by the core proteins of fibromodulin and biglycan (4). The cell surface-associated membrane chondroitin sulfate proteoglycan NG2 was originally detected in cells from the rodent central nervous system but subsequently was also detected in blood vessels and cartilaginous structures of the head, neck, and spine. It interacts via its core protein with type VI collagen and is thought to provide machinery for transmembrane signaling (5). Since ␣1␤1 and ␣2␤1 integrins also bind type VI collagen (6, 7), the cell signaling potential of this molecule woul...

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…The collagen, composed of three different subunits, was found to selfassemble through a staggered disulfide-stabilized tetramer intermediate to microfibrils (Furthmayr et al 1983;Zhang et al 2002). Type VI collagen binds under in vitro conditions to type IV collagen and type I collagen, linking the basement membrane to the adjacent stroma (Bonaldo et al 1990;Kuo et al 1997). It also binds to perlecan.…”

Congenital muscular dystrophy. Part II: a review of pathogenesis and therapeutic perspectives

“…In addition to laminin, type IV collagen is the most abundant glycoprotein in the basement membrane and all together the components of the basement membrane form a thin pericellular matrix that is involved in many cellular functions (gene expression, adhesion, migration, differentiation, and apoptosis) 64,65 . Through its carboxyl-terminal globular domain collagen IV interacts with the amino-terminal domain of the collagen VI alpha-1 chain 65 and this connection is essential for anchoring the basement membrane of the muscular fiber to the other components of the extracellular matrix and maintaining the structural muscle integrity [65][66][67][68] . Collagen VI provides a microfilamentous network in the extracellular matrix of the muscular tissue, as well as in other organs [66][67][68] .…”

Identification of a Major Microfibril-associated Glycoprotein-1-binding Domain in Fibrillin-2

“…Y2H has been used by several laboratories to document interactions between other extracellular matrix proteins, including type IV collagen (24), thrombospondin 1, the laminin ␤3 chain, and the NC1 domain of type VII collagen (25), as well as the microfibrillar protein emilin (26) and MAGP-2 (16). In our assays, Y2H provided a useful way to screen potential binding sites but was not sufficiently precise to unambiguously identify the correct binding sequence.…”

“…6B shows that only FBN2Ex (23)(24)(25) retained the ability to bind full-length MAGP-1. To assess whether the EGF domains that flank exon 24 in FBN2Ex (23)(24)(25) are required for its binding to MAGP-1, as was found in the yeast two-hybrid assay, we utilized an expression construct that encoded just Ex24. Ligand blot of bacterially expressed protein demonstrated that exon 24 retains the ability to bind MAGP-1 (Fig.…”

“…6), consistent with the Y2H results. Constructs coding for the front FBN2Ex (18 -22) and back Ex (23)(24)(25) halves of the active region were then expressed in bacterial cells, and the purified recombinant proteins were assayed by ligand blot. Fig.…”