| Two features have directed an investigation into the functional domains and cell biology of leprecan, namely, (a) its tetrapeptide KDEL and (b) four CXXXC domains.;In order to characterize leprecan's KDEL signal, it was shown that leprecan (a) colocalizes with PDI (another KDEL-containing protein); (b) colocalizes with the KDEL receptor; (c) and maintains an ER distribution in a cycloheximide (CHX) washout study. A KAAL mutant's ER distribution was abolished during a CHX washout and assembled into a CHOK1 matrix in a more pronounced manner than the wild type.;A literature search showed that a trypsin inhibitor from pumpkin seeds contained four CXXXC motifs and an active arginine residue which was shown to align with leprecan. We have shown that leprecan can bind to trypsin on an affinity column. We also developed a fluorescent assay to test the ability of leprecan peptides to inhibit the serine protease trypsin. Although the peptides failed to inhibit trypsin, it is possible that the whole protein, with its CXXXC motifs intact, is required for the inhibition.;A cell culture technique was developed to assemble in vitro basement membranes (BMs) which more closely resembles the in vivo counterpart. Although laminin, type IV collagen, BM-HSPG and BM-CSPG could be induced to assemble into the matrix under these conditions, S-laminin and leprecan could not. Leprecan's absence implies that a condition(s) necessary for its assembly into BMs is different than for that of other BM components.;We used immunohistochemistry to study the distribution of leprecan in developing BMs during rat nephrogenesis. Leprecan was not present in early BMs, but was first assembled in BMs of later comma and s-phase glomeruli. Its early distribution was not uniform and linear as for other BM components, but contained areas in the same BM which lacked staining. The staining was uniform in the adult kidney.;These findings indicate that the distribution of leprecan in basement membranes is not essential for their formation. Furthermore, leprecan's CxxxC motifs may function in serine protease inhibition or it may be play another role in the secretory pathway mediated by its KDEL tag. |
