Elongation of N-acetyllactosamine repeats in diantennary oligosaccharides.

Glycosylated [Asn22]lysozyme has been shown to contain N-acetyllactosamine repeats when expressed in chinese hamster ovary (CHO) cells. We find that the major portion of N-acetyllactosamine repeats are associated with diantennary oligosaccharides. In Lec2 CHO cells, which are deficient in sialylation, glycosylated lysozyme is synthesized with increased contents of N-acetyllactosamine repeats terminating in beta-galactosyl residues. In the Lec2 cells and the parental CHO cell line, Pro 5, only a minor portion of the oligosaccharides in lysozyme are of the triantennary type. Previously, it has been shown that the synthesis of N-acetyllactosamine repeats in Asn-linked oligosaccharides is enhanced by an increase in the activity of the elongating beta-N-acetylglucosaminyl transferase and by the synthesis of beta-1,6-linked antennae. The results with glycosylated lysozyme suggest that glycoproteins bearing diantennary oligosaccharides can contain several N-acetyllactosamine repeats and that the number of the latter can be increased by decreasing the activity of the capping sialyl transferases.

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry applied to multiple forms of lipases.

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry was used to investigate heterogeneous patterns and molecular masses of microbial lipases from Penicillium camembertii, Geotrichum candidum, and Pseudomonas sp. Mass spectral peaks of the native, glycosylated lipases from P. camembertii and G. candidum were broader than those of the corresponding deglycosylated enzymes, indicative of heterogeneous glycosylations. The broader peaks in the mass spectra were caused by an overlapping of unresolved peaks, derived from single glycoprotein species. Molecular masses determined for the deglycosylated proteins were in excellent agreement with those deduced from amino acid composition and sequence data, whereas with conventional biochemical methods (gelfiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis) only very rough estimations of molecular masses were possible. By mass spectrometric analysis of the four fractions of chromatographically separated P. camembertii lipase molecular masses of 29,990, 34,030, 31,990, and 32,140 Da were found before and 29,960, 29,980, 29,990 and 30,010 Da, respectively, after deglycosylation. Thus from the four native fractions of P. camembertii lipase three were glycoproteins. G. candidum lipase showed an average molecular mass of 63,500 Da for the heterogeneously deglycosylated native form and a molecular mass of 59,650 Da for the deglycosylated enzyme. For the Pseudomonas lipase, which could only be isolated with lipids firmly attached, a molecular mass of 32,890 Da was determined, in close agreement with that derived from the cDNA sequence.