Quantitative assessment of successive carbohydrate additions to the clustered O-glycosylation sites of IgA1 by glycosyltransferases

Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or immunoglobulin Al (IgA1). Here, we describe a series of nano-liquid chromatography (LC)-mass spectrometry (MS) analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgAl. By utilizing nano-LC-MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgAl O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrasnfersase (GaINAc-T EC 2.4.1.41) and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and alpha 2,3- and alpha 2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgAl, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgAl O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.

Automated summary

The nano-LC-MS analyses on clustered O-glycan biosynthesis on IgAl provide valuable insights into the enzymatic activities of glycosyltransferases and the effects of pre-existing glycans on subsequent activities. These findings have potential implications in understanding the molecular mechanisms involved in IgA nephropathy, and can be applied to the studies of other glycosyltransferases involved in mucin-type core 1 clustered O-glycosylation.