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Abstract
Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the alpha3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the alpha1, alpha2, alpha4, and alpha5(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 adhesion-promoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide alpha3(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 microg/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 alpha3(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 alpha3 chain and may represent an important functional epitope.
Highlights
Type IV collagen is a major component of basement membranes
Characterizations of Monoclonal Antibodies—To characterize the monoclonal antibodies that recognize a functionally important domain within the peptides of the NC1 domain of the a3(IV) chain of type IV collagen, we determined the ability of monoclonal antibodies to bind selectively to the synthetic peptides from the partial sequences of the a3(IV) chain and from the comparable region of the different a chains
Our previous studies have shown that the peptide a3(IV)185–203 prevents activation of human PMN, and the unique sequence -SNS- is the domain required for the inhibition of O22 production and enzyme release by these cells [15]
Summary
Type IV collagen is a major component of basement membranes. The predominant molecular species is a heterotrimer composed of two a1 and one a2 chain. One synthetic heparin-binding peptide, Hep-I, originating from NC1 domain of the a1 chain of type IV collagen and containing 12 amino acids, has been reported to promote the adhesion and spreading of bovine aortic endothelial cells [12, 13]. Our studies show that a synthetic peptide comprising residues 185–203 of the NC1 domain of the a3(IV) chain promotes adhesion and inhibits proliferation of melanoma cells. Using monoclonal antibodies that recognize the above peptide, we have shown that there is a multifunctional domain within the first 12 amino acids of the residue sequence 185–203 capable of promoting the cell adhesion and inhibition of proliferation. Because synthetic peptides of the same region from the other a(IV) chains that lack the triplet -SNS- (residues 189 –191) fail to inhibit melanoma cell adhesion and proliferation and because the sequence -SNS- is unique to the a3(IV) chain we can assume that this triplet represents the functional epitope of this peptide
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