Type IV Collagen of Engelbreth-Holm-Swarm Tumor Matrix: Identification of Constituent Chains

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Abstract
The noncollagenous hexamer (NCI) domain of type IV collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix was subjected to electrophoretic, amino-terminal amino acid sequence, and immunochemical analysis to determine which of the five known kinds of α(IV) chains are present. Electrophoretic analysis, whether by one-dimensional or two-dimensional electrophoresis, showed that nonlathyritic and lathyritic hexamer gave nearly identical patterns. Amino-terminal amino acid sequence analysis of hexamer subunits, transblotted from two-dimensional gels, revealed that the hexamer subunits were derived exclusively from the α 1 and α2 chains. Western blots of hexamer subunits confirmed the sequence results, as the subunits identified as α1(IV) and α2(IV) NC1 domains reacted with antibodies directed specifically against those subunits. Conversely, no reactivity of NC1 hexamer subunits was seen with Goodpasture serum, or with antibodies directed specifically against the α3, α4, and α5 NC1 domains, confirming the lack of α3, α4, and α5 chains. These results revealed that the type IV collagen component of the EHS sarcoma matrix is comprised exclusively of α1 and α2 chains. Its relative homogeneity simplifies, but restricts, interpretation of studies that employ it as a model type IV collagen because the studies would be based only on α1 and α2 chains.