Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded homodimeric mucin-like glycoprotein on leukocytes that interacts with both P- and E-selectin. In this report we describe the structures of the Ser/Thr-linked O-glycans of PSGL-1 synthesized by HL-60 cells metabolically radiolabeled with 3H-sugar precursors. In control studies, the O-glycans on CD43 (leukosialin), a mucin-like glycoprotein also expressed by HL-60 cells, were analyzed and compared to those of PSGL-1. O-Glycans were released from Ser/Thr residues by mild base/borohydride treatment of purified glycoproteins, and glycan structures were determined by a combination of techniques. In contrast to expectations, PSGL-1 is not heavily fucosylated; a majority of the O-glycans are disialylated or neutral forms of the core-2 tetrasaccharide Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAcOH++ +. A minority of the O-glycans are alpha-1,3-fucosylated that occur as two major species containing the sialyl Lewis x antigen; one species is a disialylated, monofucosylated glycan, and the other is a monosialylated, trifucosylated glycan having a polylactosamine backbone. CD43 lacks the fucosylated glycans found on PSGL-1 and is enriched for the nonfucosylated, disialylated core-2 hexasaccharide. These results demonstrate that PSGL-1 contains unique fucosylated O-glycans that are predicted to be critical for high affinity interactions between PSGL-1 and selectins.     

Phosphorylation and O-glycosylation sites of human chromogranin A (CGA79-439) from urine of patients with carcinoid tumors

Because of their water-soluble properties, chromogranins (CGs) and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived fragments have been shown to be excreted into the urine in patients with carcinoid tumors and the present study deals with the characterization of the post-translational modifications (phosphorylation and O-glycosylation) located along the largest natural CGA-derived fragment CGA79-439. Using mild proteolysis of peptidic material, high performance liquid chromatography, sequencing, and mass spectrometry analysis, six post-translational modifications were detected along the C-terminal CGA-derived fragment CGA79-439. Three O-linked glycosylation sites were located in the core of the protein on Thr163, Thr165, and Thr233, consisting in di-, tri-, and tetrasaccharides. Three phosphorylation sites were located in the middle and C-terminal domain, on serine residues Ser200, Ser252, and Ser315. These modified sites were compared with sequences of others species and discussed in relation with the post-translational modifications that we have reported previously for bovine CGA.     

Post-column enzyme reactors for chemiluminometric detection of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in an anion-exchange chromatographic system

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, from Dictyostelium discoideum. A purified contact site A was digested with Achromobacter protease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH2 terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH2 terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus in D. discoideum as observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell-cell contact regions but sparsely distributed in noncontact regions.     

N- and O-glycosylation and phosphorylation of the bar secretion leader derived from the barrier protease of Saccharomyces cerevisiae

A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha 1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure.     

The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.     

Pharmacological characterization of human m1 muscarinic acetylcholine receptors with double mutations at the junction of TM VI and the third extracellular domain

A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity. By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies. Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors. l-Hyoscyamine inhibited the activities of choline and methylcarbachol. Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp. These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.     

O-GLYCBASE version 4.0: a revised database of O-glycosylated proteins

O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been compiled from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, a literature reference and http-linked cross-references to other databases. Version 4.0 contains 179 protein entries, an approximate 15% increase over the last version. Sequence logos representing the acceptor specificity patterns for GalNAc, GlcNAc, mannosyl and xylosyl transferases are shown. The O-GLYCBASE database is available through the WWW at http://www.cbs.dtu.dk/databases/OGLYCBASE/     

Structure analysis of acetylated and non-acetylated O-linked MUC1-glycopeptides by post-source decay matrix-assisted laser desorption/ionization mass spectrometry

We have investigated the potential of structural elucidation of O-linked glycopeptides by post-source decay matrix-assisted laser desorption ionization mass spectrometry (PSD-MALDI-MS). In order to establish detailed fragmentation patterns and to dissect fragment ions with and without carbohydrate content, the same O-linked MUC1-derived glycopeptides with acetylated and non-acetylated sugars were analysed and compared. Furthermore, we were interested to examine possible differences in the fragmentation between glycopeptides with acetylated and non-acetylated sugars. The obtained PSD-MALDI-MS spectra showed a rather complete set of fragmentation data which allows to localize the glycan on the peptide, in parallel with sequencing a short glycan and the backbone peptide. Fragmentations of the sugars were dominated by inter-ring cleavages at the glycosidic bond. Intra-ring cleavage did also occur from the non-reducing end, but to a much lower extent. The fragmentation of the peptide backbone was not changed either by acetylated or non-acetylated sugars. Glycosylated peptide fragments occurred as fully glycosylated fragment ions, partially deglycosylated ions and completely deglycosylated ions, and was not influenced by the acetylation of sugars. However, differences occurred in the quality and quantity of fragment ions from the non-reducing end of the glycan part when comparing acetylated with non-acetylated glycopeptides.     

Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat

A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.     

The primary structure of caprine PP3: amino acid sequence, phosphorylation, and glycosylation of component PP3 from the proteose-peptone fraction of caprine milk

Proteose-peptone component 3 is a phosphorylated glycoprotein that was isolated from the proteose-peptone fraction of caprine milk. By mass spectrometric analysis, amino acid sequencing, and polymerase chain reaction analysis, the primary structure has been determined and has been shown to contain 136 amino acids. Phosphorylations were identified at Ser30 and Ser41. A partial glycosylation was present at Thr16, and a N-linked glycosylation was present at Asn78. Galactosamine was the amino sugar detected at Thr16. Glucosamine and galactosamine were the amino sugars found in the carbohydrate group linked to Asn78. The caprine amino acid sequence exhibits 88% identity with the bovine proteose-peptone component 3 sequence. However, when compared with the bovine sequence, the caprine sequence contains an insertion of a serine residue at position 25.     

Overview of N- and O-linked oligosaccharide structures found in various yeast species

Yeast and most higher eukaryotes utilize an evolutionarily conserved N-linked oligosaccharide biosynthetic pathway that involves the formation of a Glc3Man9GlcNAc2-PP-dolichol lipid-linked precursor, the glycan portion of which is co-translationally transferred in the endoplasmic reticulum (ER) to suitable Asn residues on nascent polypeptides. Subsequently, ER processing glycohydrolases remove the three glucoses and, with the exception of Schizosaccharomyces pombe, a single, specific mannose residue. Processing sugar transferases in the Golgi lead to the formation of core-sized structures (Hex<15GlcNac2) as well as cores with an extended poly-alpha1,6-Man 'backbone' that is derivatized with various carbohydrate side chains in a species-specific manner (Hex50-200GlnNAc2). In some cases these are short alpha1,2-linked Man chains with (Saccharomyces cerevisiae) or without (Pichia pastoris) alpha1,3-Man caps, while in other yeast (S. pombe), the side chains are alpha1,2-linked Gal, some of which are capped with beta-1,3-linked pyruvylated Gal residues. Charged groups are also found in S. cerevisiae and P. pastoris N-glycans in the form of mannose phosphate diesters. Some pathogenic yeast (Candida albicans) add poly-beta1,2-Man extension through a phosphate diester to their N-glycans, which appears involved in virulence. O-Linked glycan synthesis in yeast, unlike in animal cells where it is initiated in the Golgi using nucleotide sugars, begins in the ER by addition of a single mannose from Man-P-dolichol to selected Ser/Thr residues in newly made proteins. Once transported to the Golgi, sugar transferases add one (C. albicans) or more (P. pastoris) alpha1,2-linked mannose that may be capped with one or two alpha1,3-linked mannoses (S. cerevisiae). S. pombe is somewhat unique in that it synthesizes a family of mixed O-glycans with additional alpha1,2-linked Man and alpha1,2- and 1, 3-linked Gal residues.     

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.     

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency: structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.     

Two different glycosyltransferase defects that result in GalNAc alpha-O-peptide (Tn) expression

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.     

Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide

The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.     

Sodium lactate affects sensory and objective characteristics of tray-packed broiler chicken breast meat

We have determined the minimal portion of the retinoblastoma protein (Rb) that can serve as an efficient substrate for in vitro phosphorylation by cdk4 kinase-D1 cyclin. Kinetic measurements indicate that in vitro, a 15-kDa fragment that represents the C-terminus of Rb can serve equally well as a substrate when compared with the larger 56-kDa fragment of Rb, which contains the A, B and C domains. By comparison, peptide substrates appear to be 1000-fold less efficient. Furthermore, mutational analysis indicates that not all of the five phosphorylation sites within this minimal C domain are phosphorylated equally by cdk4/D1. Ser795 is the preferred phosphorylation site, whereas the four remaining sites Ser807, Ser811, Thr821 and Thr826 are phosphorylated to a much lesser degree. Truncations of the C domain from the carboxy terminus indicate that almost all of this domain is required for efficient phosphorylation. These data suggest that the structural context of the phosphorylation site within the substrate is critical for its phosphorylation by the cdk4/D1 kinase.