Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides

Abstract

Native type-IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a 2nd pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethylcellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange and high-performance liquid chromatography. All of the peptides had amino acid compositions characteristic of type-IV collagen. Analysis of the 8 major peptides by amino-terminal amino acid sequencing and by CnBr and tryptic peptide mapping revealed the manner in which they are derived from type-IV collagen. Pepsin liberates 2 large peptides by attacking non-triple-helical regions, one derived from the .alpha.1(IV) chain (F2, Mr 90,000) and one derived from the .alpha.2(IV) chain (F3, Mr 75,000). The .alpha.1(IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments (F4c [Mr 41,000] and F4a [Mr 60,000]), as is the .alpha.2(IV)-derived F3 peptide (F5 [Mr 28,000] and F4b [Mr 50,000], respectively). Apparently, the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. Several of the peptides examined were present in 2 slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type-IV collagen molecules. These methods provide a reliable means by which identifiable type-IV collagen peptides can be isolated. The above conclusions and data provide a basis from which the previously described type-IV collagen peptides can be more clearly identified and related to a common structure of origin.