Yeast Kre1p is a cell surface O-glycoprotein

The Saccharomyces cerevisiae KRE1 gene encodes a secretory protein required for the production of the cell wall polymer (1 6)--glucan. Here we report further characterization of the KRE1 gene product, Krelp. A functional, epitope-tagged Krelp is shown to be highly modified in a SEC53-dependent manner. Krelp is O-glycosylated, but the basis for the majority of its post-translational modification is unknown. Fractionation of Kre1p reveals a cell wall-associated form and a less abundant membrane-associated species. Indirect immunoflurorescence demonstrates that Kre1p localizes to the cell surface, where it becomes concentrated at the surface of mother cells. Such a localization of Kre1p seems to parallel the CAL1/CSD2-dependent cell wall deposition of chitin found in S. cerevisiae, and is consistent with evidence from Schizophyllum commune that (1 6)--glucan accumulates during maturation of the subapical region of the wall distal to the hyphal tip.     

Tau protein kinase I/GSK-3Β/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain

Previously, tau protein kinase I/glycogen synthase kinase-3/kinase F_A(TPKI/GSK-3/F_A) was identified as a brain microtubule-associated tau kinase possibly involved in the Alzheimer disease-like phosphorylation of tau. In this report, we find that the TPKI/GSK-3/F_A can be stimulated to phosphorylate brain tau up to 8.5 mol of phosphates per mol of protein by heparin, a polyanion compound. Tryptic digestion of³²P-labeled tau followed by high-performance liquid chromatography and high-voltage electrophoresis/thin-layer chromatography reveals 12 phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and peptide sequence analysis further reveals that TPKI/GSK-3//F_A after heparin potentiation phosphorylates tau on sites of Ser¹⁹⁹, Thr²³¹, Ser²³⁵, Ser²⁶², Ser³⁹⁶, and Ser⁴⁰⁰, which are potential sites abnormally phosphorylated in Alzheimer tau and potent sites responsible for reducing microtubule binding possibly involved in neuronal degeneration. The results provide initial evidence that TPKI/GSK-3/F_A after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau and neuronal degeneration in Alzheimer disease brains.Abbreviations F_A the activating factor of type 1 protein phosphatase - GSK-3 glycogen synthase kinase-3 - TPKI tau protein kinase I - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - PHF paired helical filaments - HPLC high-performance liquid chromatography     

FAB CID-MS/MS characterization of tetrasaccharide tri- and tetrasulfate derived from the antigenic determinant recognized by the anti-chondroitin sulfate monoclonal antibody MO-225

The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MS/MS mass spectrometry/mass spectrometry - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose     

Functional domains of bovine β-1,4 galactosyltransferase

A number of N- and C-terminal deletion and point mutants of bovine -1,4 galactosyltransferase (-1,4GT) were expressed inE. coli to determine the binding regions of the enzyme that interact withN-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1–129, do not show any significant difference in the apparentK _ms toward NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15mm NAG and 50mm EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130–402 of bovine -1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130–257 of the -1,4GT, binds to, and elutes with 15mm NAG and 50mm EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15mm NAG. The C-terminus fragment GT-257UDP, containing residues 258–402 of -1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5–10% of the bound protein, can be eluted from the UDP-agarose column with 50mm EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of -1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn²⁺.     

Glycoforms of serum α1-acid glycoprotein as markers of inflammation and cancer

1-acid glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked complex type heteroglycan side chains which may be present as bi-, tri- and tetraantennary structures. Depending upon the content of biantennary structure on AGP, up to four glycoforms of AGP are present in serum. These glycoforms can be easily estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the more biantennary structures on AGP, the stronger the binding. In acute inflammation, a relative increase of AGP glycoforms with biantennary units is observed - a type I glycosylation change. In some chronic inflammatory states there is an relative decrease of AGP glycoforms with biantennary heteroglycans — a type II glycosylation change. Moreover, in certain other states such as pregnancy, estrogen administration or liver damage, type II glycosylation changes are also seen. A detailed analysis of the clinical applications of the assessment of AGP glycoforms in sera of patients with rheumatic diseases, AIDS and various types of cancers is presented. Accumulated data shows that AGP glycoforms may be very useful in the detection of intercurrent infections in the course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic leukaemia, and in the detection of secondary infections in human immunodeficiency virus infected individuals. AGP glycoforms are also very useful in differentiation between various forms of trophoblastic disease and are helpful in monitoring the treatment of these patients. Finally, AGP glycoforms provide valuable information for differentiation between primary and secondary liver cancer.     

Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of the cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRAPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type I turn involving the same amino acids in both glycosylated and unglycosylated peptides. The GalNAc attached to the threonine (T³) and serine (S⁴) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GM2 monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline