The distribution, deposition and stabilization of different collagen types are crucial for the formation and maintenance of functional connective tissue matrices. We plan to investigate the extent and importance of aldehyde-derived and disulfide-derived crosslinks between the same and different collagen types during tissue development and maturation. We will use both in vitro and in vivo models of fibrillogenesis to study the location and temporal sequence of crosslink formation. These data will identify collagen packing arrangements in fibrillar and non-fibrillar extracellular matrices. Collagen fibers will be reconstitiuted from type I and/or type III collagen using mixtures of labeled lathyritic and unlabeled normal collagen. This will permit the identification of amino and aldehyde donors in crosslink formation. The frequency and location of crosslinks along different kinds of collagen chains will be determined by collagenase digestion and 2-D CNBr mapping. The diversity of crosslink patterns between homopolymers and heteropolymers in skin, bone, tendon and gramulation tissues will be explored using methods to identify and purify crosslinked peptides containing the carboxy-terminal CNBr peptides of type I and type III collagen. This will be accomplished by using monoclonal antibodies to a1(I)-CB6 and a2(I)-CB5, and a sulfhydryl affinity column for a1(III)-CB9. These peptides will be further fractionated by 2-D CNB4r mapping and then characterized by tryptic fingerprinting and amino acid analysis. The presence and formation of intermolecular disulfide crosslinks in type III collagen has been demonstrated in our laboratory. We will test whether these crosslinks allow type III collagen to form a transient early scaffolding for developing tisuses by identifying type III disulfide bonded polymers during formation of granulation tissue. Polymers will be identified by sedimentation and 2-D mapping. Finally, the structure of EC collagen will be investigated with emphasis on the sequence and polarity of the peptides. The molecular interactions of EC collagen in basement membrane architecture will be analyzed by determining the location of crosslinked CNBr peptides beteen itself and type IV collagen. Crosslinked peptides in Descemet's membrane an basement membrane derived from cultured corneal endothelial cells will be labelled, fractioned by 2-D mapping and characterized by tryptic fingerprinting on HPLC.
