We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.

This study shows for the first time that different glycosyltransferase defects in the biosynthesis of O-linked oligosaccharides give rise to the same GalNAc alpha-O-Ser/Thr determinant on Tn erythrocytes and colorectal carcinoma cells. The O-linked oligosaccharides isolated from the glycophorins of Tn erythrocytes contained predominantly alpha-N-acetylgalactosamine-O-Ser/Thr (Tn antigen) and sialyl-Tn. A marked reduction in normal sialylated oligosaccharides was also observed. Monoclonal antibody BRIC 111 raised against Tn erythrocytes reacted with both Tn erythrocytes and colorectal carcinoma tissues. Weak staining was detected in the supranuclear area and at the surface membranes in normal colorectal cells, but was absent from goblet cell vesicles. An increase in supranuclear staining over controls was found in tumour tissue and in the majority of resection margin specimens. The highest levels of staining were present in transitional mucosa, adjacent to the tumours where goblet vesicles were also positive. Glycosylation defects in the same patients were further studied by determination of the activity of glycosyltransferases in mucosal tissue from control and cancer patients. The reduction in or loss of beta 1-3 N-acetylglucosaminyl transferase activity to GalNAc-peptide in asialo-ovine submaxillary gland glycoprotein was detected by direct assay and by isolation of the oligosaccharides from the incubation products. No differences in N-acetylglucosaminyl-, galactosyl- or sialyl-transfer to Gal beta 1-3GalNAc in antifreeze glycoprotein or in sialyl transferase to asialo-ovine submaxillary gland glycoprotein were detected. Our study shows that the GalNAc alpha-O-Ser/Thr determinant on Tn erythrocytes and in colorectal carcinoma results from different glycosyltransferase defects in separate biosynthetic pathways for haematopoietic and epithelial tissues.

No abstract

Immunoprecipitation is a traditional approach to isolate single proteins or native protein complexes from a complex sample mixture. The original method makes use of specific antibodies against endogenous proteins or epitope tags, which are first bound to the target protein and then isolated with protein A beads. An advancement of this method is the application of a protein A tag fused to the target protein and the affinity-purification of the tagged protein with human Immunoglobulin G chemically cross-linked to a sepharose matrix. This method will be described exemplified by the purification of protein complexes of the peroxisomal membrane from yeast Saccharomyces cerevisiae.