The O-glycosylation of mucin-type molecules is responsible for their unique physicochemical properties. Despite the fact that O- glycosylation is ubiquitous, little is known about the way it is regulated. We are studying the initiation of glycosylation by UDP- N-acetylgalactosamine:peptide N-acetylgalactosamine transferase. We have formed an extensive database of known glycosylated sites and their flanking sequences. Computer analysis of the amino acid sequences surrounding glycosylated site does not reveal a strict consensus, but suggests that sequence may influence the initiation of O-glycosylation, particularly at positions -1 and +3, relative to the glycosylated site. A series of test peptides was made to experimentally verify this hypothesis. It was found that single amino acid changes at the 'sensitive' positions near a threonine residue could dramatically alter its ability to be glycosylated. Furthermore, circular dichroism spectroscopy showed that all the substrates that were glycosylated had random-type structure. A set of 50 peptides has been synthesized to extend the investigation of flanking sequences. Serine-containing substrates glycosylate poorly compared to those containing threonine. GalNAc transferase was partially purified from a number of rat, human and bovine tissue. Assays of serine- containing peptides reveals that their inability to be glycosylated in vitro is not due to flanking sequence alone.. We have suggested that a separate GalNAc transferase is responsible for glycosylating serine, and are currently attempting to isolate this activity. It seems possible that a family of GalNAc transferases exists, having multiple overlapping sequence specificities. This would explain the absence of a single consensus sequence for all glycosylated sites. Future studies, involving the design and production of recombinant mucin-like species (eg for replacement therapy in salivary hypofunction) will have to take into account both substrate sequence requirements and enzyme activities available to successfully facilitate their glycosylation.
Showing the most recent 10 out of 22 publications
