| Collagen IV networks are present in all metazoan and underlie epithelia as a component of basement membranes (BM). The networks are essential for tissue function and are defective in disease. They are assembled by the oligomerization of triple-helical protomers that are linked end-to-end. At the C-terminus, two protomers are linked head-to-head by interactions of their trimeric noncollagenous domains, forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce the quaternary structure of hexamer. Recently, the crystal structure of the NC1 hexamer ruled out the covalent stabilization by disulfide reshuffling across the trimer-trimer interface, which had been accepted for more than twenty years. Alternatively, the x-ray crystallography studies at 1.9 A resolution of placenta BM suggested a putative covalent crosslink between the side chains of Met 93 and Lys211 of opposing NC1 trimers (Than et. al. 2002). However, the exact chemical structure of the crosslink remained undetermined.; Therefore, the goal of this dissertation was to elucidate the location and chemical nature of the crosslinks that stabilize the NC1 hexamer. The study was undertaken by using a traditional protein chemistry approach in combination with the sophisticated structural techniques of x-ray protein crystallography and mass spectrometry. The main conclusions of the combined strategy are: (1) two kinds of NC1 hexamers exist; M-hexamers and D-hexamers composed exclusively of monomers and dimers, respectively; (2) Hydroxylysine (Hyl211 exists in the NC1 domain of collagen IV molecules; and (3) the NC1 hexamer is stabilized by a crosslink involving Hyl 211 and Met93, termed S-hydroxylysyl-methionine crosslink. Since hydroxylysine is normally glycosylated, a mechanism where carbohydrates regulate the interaction between collagen IV molecules is suggested. These findings not only correct a long standing misconception about the crosslinking of collagen IV, but they also provide significant chemical evidence to motivate the search for an unknown/putative enzyme which assemble the crosslink. |
