The Function of the NC1 Domains in Type IV Collagen

191

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (␣1 to ␣5) of the six chains of type IV collagen, organized into an ␣1⅐␣2(IV) and an ␣3⅐␣4⅐␣5(IV) network. In Alport syndrome, mutations in any of the genes encoding the ␣3(IV), ␣4(IV), and ␣5(IV) chains cause the absence of the ␣3⅐␣4⅐␣5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the ␣5 chain was shown to be linked to the ␣3 and ␣4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified ␣1-␣5(IV) NC1 monomers. The results showed that NC1 monomers associate to form nativelike hexamers characterized by two distinct populations, an ␣1⅐␣2 and ␣3⅐␣4⅐␣5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the ␣1⅐␣2 and ␣3⅐␣4⅐␣5 networks of GBM. Moreover, hexamer formation from the ␣3, ␣4, and ␣5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the ␣3⅐␣4⅐␣5 network by interfering with the assembly of the ␣3⅐␣4⅐␣5 NC1 hexamer.

Section: Discussionmentioning

confidence: 98%

Type IV Collagen of the Glomerular Basement Membrane

Boutaud¹,

Borza²,

Bondar³

et al. 2000

191

“…Polyclonal and monoclonal (M3F7) type IV collagen antibodies were from Southern Biotechnology Associates, Inc. and the Developmental Studies Hybridoma Bank (University of Iowa), respectively. Goat anti-mouse 5-nm colloidal gold conjugate was from Amersham Corp. Polyclonal antibodies against ␣1(IV) NC1 and ␣2(IV) NC1 were gifts from Klaus Kü hn (Martinsried, Munich) (19). The type VI collagen monoclonal antibodies N3-VI-2, C1-VI-1, and C12-VI-2 have been described previously (7).…”

Section: Methodsmentioning

confidence: 99%

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

Kuo

Maslen

Keene

et al. 1997

297

215

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...

“…The NC1 domain is thought to play a key role in the alignment and selection of three ␣ chains forming a triple helical molecule (10,11). Several studies have focused on the biological activity of the NC1 domain of type IV collagen.…”

mentioning

confidence: 99%

A Cell Binding Domain from the α3 Chain of Type IV Collagen Inhibits Proliferation of Melanoma Cells

Han¹,

Ohno²,

Brassart‐Pasco³

et al. 1997

Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the ␣3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the ␣1, ␣2, ␣4, and ␣5(IV) chains did not exhibit this property. Because of the intimate relationship between metastasizing neoplastic cells and vascular as well as epithelial basement membranes, we measured the cell adhesionpromoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide ␣3(IV)185-203 (CNYYSNSYSFWLASLNPER) not only promotes adhesion of human melanoma cells but also inhibits their proliferation. Adhesion increased by 50 -60% over control. Melanoma cell proliferation was inhibited by 40% when cells were grown in a medium containing 5 g/ml peptide for 5 days. Studies showed that replacement of serine in position 189 or 191 by alanine resulted in significantly reduced adhesion. Similarly, serine replacement resulted in reduced ability to inhibit proliferation. Our data suggest that a region of the NC1 domain of the ␣3(IV) chain, contained within the sequence 185-203, not only specifically promotes adhesion but also inhibits proliferation of melanoma cells. These properties appear to be dependent on the presence of the triplet sequence -SNS-(residues 189 -191), which is unique to the ␣3 chain and may represent an important functional epitope.