We have isolated a large peptide from human amniotic membrane which contains two major structural domains. Approximately 90% of the molecule has the characteristics of a collagen triple helix. The remaining 10% of the structure appears to be nontriple helical, and is distributed at both ends and in a central region within the triple helix. Because of this discontinuity in the triple helix, the molecule is extremely sensitive to pepsin proteolysis. The intact molecule was isolated by modifying the pepsin-solubilization procedures normally used for collagen isolation to prevent excessive proteolysis. When compared with other human collagens, the amino acid composition, the electrophoretic profiles of fragmentation products generated by cyanogen bcromide cleavage or V8 protease digestion, and comparisons of the limit-pepsin digest product indicate that this molecule is unlike and collagen currently described. Preliminary estimates of the molecular weight of this collagen indicate that the molecule is approximately twice the length of a Type I collagen and 150% of the length of Type IV collagen. This unexpected result suggests that this molecule represents the first of a new class of collagens. Preliminary localization studies using hybridoma technology suggest that the molecule is associated with the amniotic epithelial basement membrane. We propose to purify this new collagen to homogeneity to document its composition of amino acids and neutral hexose, and to compare its structural features with other described collagens. We intend to isolate and characterize the triple-helical and nontriple-helical domains of the molecule, and to assess order along the length of this new collagen. Although this molecule is substantially larger than previously described collagens, it is a proteolytic fragment of a larger matrix component. To assess the structure of the parent molecule, we will examine the precursor form of this molecule synthesized by amniotic epithelial cells in culture. We will produce monoclonal antibodies to this collagen and determine its matrix location relative to the amniotic epithelial cells and the underlying basement membrane by immunolocalization techniques at the light and electron microscopic level. This study will generate immunological probes for the assessment of a possible role for this molecule in selected human disease conditions involving basement membrane dysmorphology.
NIH ExPorter
NIH ExPorter