Biomedical subjects

H Paulsen

Publications and source records attributed to H Paulsen.

At least 55 records · Page 3Linked to original sources

PubMed1995

N-proximal sequence motif in light-harvesting chlorophyll a/b-binding protein is essential for the trimerization of light-harvesting chlorophyll a/b complex.

The major light-harvesting complex (LHCII) of photosystem II can be reconstituted in its native, trimeric form starting from its apoprotein light-harvesting chlorophyll a/b-binding protein (LHCP), pigments, and thylakoid lipids. In this paper we identify segments in the LHCP polypeptide that are essential for the formation of stable LHCII trimers by analyzing N- and C-terminal deletion mutants of LHCP and mutants carrying point-specific amino acid exchanges. C-terminal deletions that do not abolish pigment binding to LHCP do not affect trimerization either. By contrast, on the N-terminus of LHCP, where as many as 61 amino acids can be deleted without significant effects on pigment binding, only 15 amino acids are dispensible for LHCII trimer formation. This indicates that structural elements between amino acids 16 and 61 are involved in the stabilization of LHCII trimers but not monomers. Closer inspection of this protein domain in a more detailed mutation analysis revealed that amino acids W16 and/or Y17 as well as R21 are essential for the formation of LHCII trimers. These amino acids are conserved in virtually all known sequences of LHCII apoproteins but only in some of the minor chlorophyll a/b complexes. Possible functions of the crucial residues are discussed.

Amino Acid Sequence↗

PubMed1995

Synthesis of the glycosyl amino acids N alpha-Fmoc-Ser[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-OPfp and N alpha-Fmoc-Thr[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-OPfp and the application in the solid-phase peptide synthesis of multiply glycosylated mucin peptides with Tn and T antigenic structures.

Two new glycosyl amino acids N alpha-Fmoc-Ser[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-+ ++OPfp and N alpha-Fmoc-Thr[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-+ ++OPfp were synthesized. Glycosylation of N alpha-Fmoc-Ser-OPfp or N alpha-Fmoc-Thr-OPfp with protected beta-D-Gal-(1-->3)-D-GalN3 donors afforded the glycosyl amino acids containing an activated C-terminus which could be utilized directly for solid-phase glycopeptide synthesis. The transformation of the 2-azido group into the acetamido derivative was achieved quantitatively at the end of the synthesis by treatment of the polymer-bound glycopeptide with thioacetic acid. The versatility of this strategy was demonstrated by the assembly of eight triply glycosylated mucin peptides which were synthesized simultaneously by multiple column techniques. The glycopeptides were prepared in order to investigate the substrate specificity of a galactosyltransferase.

Amino Acid Sequence↗

PubMed1995

Synthetic substrate analogues for UDP-GlcNAc: Man alpha 1-3R beta 1-2-N-acetylglucosaminyltransferase I. Substrate specificity and inhibitors for the enzyme.

UDP-GlcNAc:Man alpha 1-3R beta 1-2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) catalyses the conversion of [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6][Man alpha 1-3]Man beta-O-R to [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6] [GlcNAc beta 1-2Man alpha 1-3]Man beta-O-R (R = 1-4GlcNAc beta 1-4GlcNAc- Asn-X) and thereby controls the conversion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-T I also catalyses the conversion of Man alpha 1-6(Man alpha 1-3)Man beta-O-octyl to Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta-O-octyl. We have therefore tested a series of synthetic analogues of Man"alpha 1-6(Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T I. The 2"-deoxy and the 3"-, 4"- and 6"-O-methyl derivatives are all good substrates confirming previous observations that the hydroxyl groups of the Man"alpha 1-6 residue do not play major roles in the binding of substrate to enzyme. In contrasts, all four hydroxyl groups on the Man'alpha 1-3 residue are essential since the corresponding deoxy derivatives either do not bind (2'- and 3'-deoxy) or bind very poorly (4'- and 6'-deoxy) to the enzyme. The 2'- and 3'-O-methyl derivatives also do not bind to the enzyme. However, the 4'-O-methyl derivative is a substrate (KM = 2.6 mM) and the 6'-O-methyl compound is a competitive inhibitor (Ki = 0.76 mM). We have therefore synthesized various 4'- and 6'-O-alkyl derivatives, some with reactive groups attached to an O-pentyl spacer, and tested these compounds as reversible and irreversible inhibitors of GlcNAc-T I. The 6'-O-(5-iodoacetamido-pentyl) compound is a specific time dependent inhibitor of the enzyme. Four other 6'-O-alkyl compounds showed competitive inhibition while the remaining compounds showed little or no binding indicating that the electronic properties of the attached O-pentyl groups influence binding.

Animals↗

PubMed1995

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R beta 1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues.

UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R (GlcNAc to Man) beta 1,6- N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc beta 1-6 branch to bi- and triantennary N-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc beta 1-2Man alpha 1-6Glc/Man beta-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man alpha 1-3 residue and the 4-hydroxyl of the Man beta- residue of the Man alpha 1-6(Man alpha 1-3)Man beta-R N-glycan core are not essential for catalysis but influence substrate binding. GlcNAc beta 1-2(4,6-di-O-methyl-)Man alpha 1-6Glc beta-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Animals↗

PubMed1995

Synthesis of O-glycan core 3: characterization of UDP-GlcNAc: GalNAc-R beta 3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines.

UDP-GlcNAc: GalNAc-R beta 3-GlcNAc-transferase (core 3 beta 3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine, GalNAc is N-acetyl-D-galactosamine and T is transferase) is expressed in a tissue-specific fashion and is high in normal colonic tissue, but downregulated in colon cancer. To further study the control of this enzyme, we examined the activity in pig, rat and human colonic tissues, and several human cancer cell lines. The enzyme was difficult to solubilize by detergents and was extremely unstable in the solubilized form. Using synthetic derivatives of the GalNAc-R substrate, we showed that the specificity of the enzyme in normal rat and human colonic mucosa requires all the substituents of the GalNAc-sugar ring of substrates for maximal activity. Core 3 beta 3-GlcNAc-T was significantly influenced by the structure of the aglycon group. None of the inactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine alpha-benzyl was a weak substrate and significantly inhibited the incorporation of GLcNAc into GalNAc alpha-benzyl by human colonic homogenates. Surprisingly, none of the colonic cancer cell lines or any other cancer and leukaemia cells examined exhibited detectable activity of the enzyme, although a number of other glycosyltransferase activities involved in O-glycan biosynthesis were active. Mixing experiments did not reveal an endogenous inhibitor in HL60 cells or an activator of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T activity in cancer cell lines may be due to cell transformation or cell culturing.

Amino Acid Sequence↗

PubMed1995

Characterization of a novel mucin sulphotransferase activity synthesizing sulphated O-glycan core 1,3-sulphate-Gal beta 1-3GalNAc alpha-R.

A novel sulphotransferase (sulpho-T) activity from rat colonic mucosa was characterized using O-glycan core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl. Derivatives of Gal beta 1-3GalNAc- were used to demonstrate that the 3- and 4-hydroxyl of Gal and the 2-acetamido group of the GalNAc residue of Gal beta 1-3GalNAc alpha-benzyl substrates were important for activity. Sulphated product using Gal beta 1-3GalNAc alpha-benzyl as substrate was analysed by ion spray mass spectrometry, methylation analysis, high-pH anion-exchange chromatography and beta-galactosidase digestion. The results suggested that sulphate was added to the 3-position of the Gal residue. The synthesis of core 2 from core 1 by UDP-GlcNAc: Gal beta 1-3GalNAc beta 6-GlcNAc-transferase was inhibited by sulphation of the Gal residue, indicating that GlcNAc beta 1-6 branching has to precede sulphation in the O-glycan core 1 processing pathway. These data demonstrate several novel pathways in the synthesis of sulphated mucin-type oligosaccharides.

Animals↗

PubMed1995

Light-harvesting chlorophyll a/b-binding protein inserted into isolated thylakoids binds pigments and is assembled into trimeric light-harvesting complex.

The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem II (LHCII) but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCII and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCII monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.

Amino Acid Sequence↗

PubMed1994

Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex.

The major light-harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll-containing complex in higher plants, is organized in trimers. In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids. LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes. These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids. The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients. Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment-pigment interactions or alters pre-existing interactions. Reconstituted LHCII trimers readily form two-dimensional crystals that appear to be identical to crystals of the native complex.

Amino Acid Sequence↗

PubMed1994

Synthesis of tetrasaccharide analogues of the N-glycan substrate of beta-(1-->2)-N-acetylglucosaminyltransferase II using trisaccharide precursors and recombinant beta-(1-->2)-N-acetylglucosaminyltransferase I.

Recombinant rabbit UDP-GlcNAc: alpha-Man-(1-->3R) beta-(1-->2)-N-acetylglucosaminyl-transferase I (EC 2.4.1.101, GlcNAc-T I) produced in the Sf9 insect cell/baculovirus expression system has been used to convert compounds of the form 3-R-alpha-Man(1-->6)(alpha-Man(1-->3)) beta-Man-O-octyl to 3-R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3)) beta-Man-O-octyl where R is OH (14), O-methyl (17), O-pentyl (18), O-(4,4-azo)pentyl (19), O-(5-iodoacetamido)pentyl (20) and O-(5-amino)pentyl (21); 2-deoxy-alpha-Man(1-->6)(beta-GlcNAc(1-->2) alpha-Man(1-->3)) beta-Man-O-octyl (16), 4-O-methyl-alpha-Man(1-->6) (beta-GlcNAc(1-->2) alpha-Man(1-->3)) beta-Man-O-octyl (22), 6-O-methyl-alpha-Man(1-->6)(beta-GlcNAc(1-->2) alpha Man(1-->3)) beta-Man-O-octyl (23) and alpha-Man(1-->6)[beta-GlcNAc(1-->2)(4-O-methyl) alpha-Man(1-->3)] beta-Man-O-octyl (15) were also synthesized by this procedure. The yields ranged from 80 to 99%. Products were characterized by high resolution 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 14, 15, 17, 22, and 23 are excellent substrates for UDP-GlcNAc: alpha-Man(1-->6R) beta-(1-->2)-N-acetylglucosaminyltransferase II and the other compounds are inhibitors of this enzyme.

Animals↗

PubMed1994

UDPgalactose:glycoprotein-N-acetyl-D-galactosamine 3-beta-D-galactosyltransferase activity synthesizing O-glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates.

In order to investigate the role of the peptide moiety of glycoproteins in the control of O-glycan biosynthesis, UDPgalactose:glycoprotein-N-acetyl-D-galactosamine 3-beta-D-galactosyltransferase (core 1 beta 3-Gal-T) from rat liver was tested for its specificity towards GalNAc-containing glycopeptide substrates. Series of glycopeptides have been synthesized by solid-phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based on variations of the repeat sequences of human intestinal mucin. Most glycopeptides were excellent substrates for core 1 beta 3-Gal-T compared to benzyl alpha-D-galactosamine as indicated by their relatively high Vmax/Km. The enzyme preferred threonine alpha-D-galactosamine Thr(GalNAc) to serine alpha-D-galactosamine. Pro on the carboxy-terminal side adjacent to Thr(GalNAc) was inhibitory. Negatively charged amino acids on either side showed a low Km; substrates with negatively charged amino acids on the amino-terminal side were highly efficient substrates, suggesting charge-charge interactions between enzyme and substrate. Gal beta 1-3GalNAc alpha residues adjacent to Thr(GalNAc) reduced the activity. Product analysis using glycopeptide substrates with three adjacent GalNAc residues showed incorporation of one, two and a small amount of three Gal residues per molecule with an uneven distribution of the potential di-galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the glycosylation pathways of O-glycans is also controlled by the structure and glycosylation of the peptide core of substrates.

Amino Acid Sequence↗

PubMed1994

Synthetic substrate analogues for UDP-GlcNAc: Man alpha 1-6R beta(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme.

UDP-GlcNAc:Man alpha 1-6R beta(1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complex N-glycans. We have tested a series of synthetic analogues of the substrate Man"'alpha 1-6(GlcNAc"beta 1-2Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attaches N-acetylglucosamine in beta 1-2 linkage to the 2"'-OH of the Man"'alpha 1-6 residue. The 2"'-deoxy analogue is a competitive inhibitor (Ki = 0.13 mM). The 2"'-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3"'-, 4"'- and 6"'-OH groups are not essential for binding or catalysis since the 3"'-, 4"'- and 6"'-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3"'-position to pentyl and substituted pentyl groups causes competitive inhibition (Ki = 1.0-2.5 mM). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3"'-O-(4,4-azo)pentyl group and a 3"'-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man"'alpha 1-6 residue are essential for binding although the 2"'- and 3"'-OH face the catalytic site of the enzyme. The 4-OH group of the Man beta-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Susceptibility of glycans to beta-elimination in Fmoc-based O-glycopeptide synthesis.

In order to investigate the possible extent of beta-elimination occurring in Fmoc-based continuous-flow solid-phase glycopeptide synthesis, the influence of the pKb of the base used for N alpha-deprotection has been studied. A glycosylated pentapeptide was synthesized using 50% morpholine, 10% piperidine or 2% DBU, respectively, in DMF for deprotection. The dehydropentapeptide N alpha-Ac-Thr-Thr-delta Aba-Val-Thr-NH2, which would be formed in the case of beta-elimination, was prepared independently and used as a control in HPLC analysis; however, this product was not formed under any of the deprotection conditions applied. Furthermore, a 23 amino acid long glycopeptide from human intestinal mucin was prepared using 2% DBU as a base for Fmoc cleavage, and similarly no beta-elimination was observed. The glycopeptide products were subjected to a prolonged treatment with sodium hydroxide in methanol/water without significant formation of byproducts, and the pure glycopeptides were isolated and characterized by 1H-NMR spectroscopy.

Amino Acid Sequence↗

PubMed1993

Pigments induce folding of light-harvesting chlorophyll a/b-binding protein.

The conformational behaviour of the light-harvesting chlorophyll a/b-binding protein (LHCP), the apoprotein of the major light-harvesting complex (LHCII) of photosystem II in plants, has been studied. According to the circular dichroism in the ultraviolet range measured with isolated LHCII, the protein in the complex adopts a folded structure with a high content of alpha helix (about 60%), whereas the non-pigmented, solubilized protein has a less ordered structure (about 20% alpha helix). LHCP-pigment complexes that have been reconstituted from the overexpressed protein and isolated pigments in the presence of detergents display a protein CD signal similar to that of authentic LHCII, indicating that LHCP folds into the native structure during the reconstitution procedure. Renaturation of LHCP in these experiments is dependent on the presence of pigments and the formation of stable LHCP-pigment complexes. Pigment-induced engagement of LHCP in a compact structure has also been shown by two additional experimental approaches. (a) Upon complex formation, LHCP or its precursor (pLHCP) becomes resistant to trypsin digestion with the exception of an N-terminal segment of the protein; the same protection of LHCP is known to occur in intact thylakoids. (b) Pigment binding renders a cysteine residue within the N-proximal hydrophobic domain of the protein as well as a newly introduced cysteine four amino acid positions from the C terminus inaccessible to modification with a sulfhydryl-specific label whereas the N terminus stays susceptible to specific labelling. These observations support the notion that only the N terminus protrudes from a compact protein-pigment structure in LHCII. The fact that the major part of LHCP is trypsin-resistant in pigmented complexes reconstituted in the absence of a membrane or even lipids justifies caution in using protection against trypsin as a criterion for the integration of LHCP into the thylakoid membrane.

Apoproteins↗

PubMed1993

Processing O-glycan core 1, Gal beta 1-3GalNAc alpha-R. Specificities of core 2, UDP-GlcNAc: Gal beta 1-3 GalNAc-R(GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase and CMP-sialic acid: Gal beta 1-3GalNAc-R alpha 3-sialyltransferase.

To elucidate control mechanisms of O-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Gal beta 1-3GalNAc-R(GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (EC 2.4.1.102; core 2 beta 6-GlcNAc-T) and CMP-sialic acid: Gal beta 1-3GalNAc-R alpha 3-sialyltransferase (EC 2.4.99.4; alpha 3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 beta 6-GlcNAc-T activity; core 2 beta 6-GlcNAc-T from mucin secreting tissue (named core 2 beta 6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAc beta 1-6(GlcNAc beta 1-3)GalNAc-R] and blood group I [GlcNAc beta 1-6(GlcNAc beta 1-3)Gal beta-R] branches; core 2 beta 6-GlcNAc-T in leukemic cells (named core 2 beta-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 beta 6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls of N-acetylgalactosamine and the 6-hydroxyl of galactose of the Gal beta 1-3GalNAc alpha-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. alpha 3-sialyltransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Gal beta 1-3GalNAc alpha-Bn. Gal beta 1-3(6-deoxy)GalNAc alpha-Bn and 3-deoxy-Gal beta 1-3GalNAc alpha-Bn competitively inhibited core 2 beta 6-GlcNAc-T and alpha 3-sialyltransferase activities, respectively.

Animals↗

PubMed1993

Pigment complexes of light-harvesting chlorophyll a/b binding protein are stabilized by a segment in the carboxyterminal hydrophilic domain of the protein.

In order to identify segments of light-harvesting chlorophyll a/b-binding protein (LHCP) that are important for pigment binding, we have tested various LHCP mutants regarding their ability to form stable pigment-protein complexes in an in vitro reconstitution assay. Deletion of 10 C-terminal amino acids in the LHCP precursor, pLHCP, did not significantly affect pigment binding, whereas deletion of one additional amino acid, a tryptophan, completely abolished the formation of stable pigment-protein complexes. This tryptophan, however, can be exchanged with other amino acids in full-length pLHCP without noticeably altering the stability or spectroscopic properties of pigment complexes made with these mutants. Thus, the tryptophan residue is not likely to be involved in a highly specific interaction stabilizing the complex. A double mutant of LHCP lacking 66 N-terminal and 6 C-terminal amino acids still forms pigmented complexes that are virtually identical to those formed with the full-length protein concerning their pigment composition and spectroscopic properties. We conclude that about 30% of the polypeptide chain in LHCP is not involved in pigment binding.

Amino Acid Sequence↗

PubMed1993

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota R4 (chemotype Rd2P-)

Rabbit polyclonal antibodies against the rough mutant lipopolysaccharide (LPS) of Salmonella minnesota R4 (chemotype Rd2P-) were serologically characterized by using R4 LPS, deacylated LPS, dephosphorylated LPS, and synthetic partial structures, including compounds comprising the core region of Rd2P- LPS bound to the beta 1-->6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or coupled to bovine serum albumin. By using a passive hemolysis assay and an enzyme immunoassay and absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core oligosaccharide (with or without the side chain 3-deoxy-D-manno-octulosonic acid [Kdo]) and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The phosphate-independent antibodies were directed against the core oligosaccharide, recognizing an epitope consisting of one terminal heptose linked to Kdo or to the reducing moiety of the alpha 2-->4-linked Kdo disaccharide. Antibodies requiring the presence of acyl residues and those reacting with a single heptose or Kdo residue were not detected.

Animals↗

PubMed1992

[Synthesis of modified oligosaccharides of the N-glycoprotein as substrate for N-acetylglucosaminyltransferase I].

In the synthesis of modified derivatives of octyl O-(alpha-D-mannopyranosyl)-(1-->3)-O-[(alpha-D-mannopyranosyl)-(1-->6)]- beta-D-mannopyranoside, 4.,5-epoxypentyl, a 4-diazirinopentyl, and a 5-(iodoacetamido)pentyl group were attached to the 3''-OH of the trisaccharide. The diazirino derivative may be especially suitable for photolabeling of the active site of N-acetylglucosaminyltransferase I (GlcNAcT-I). In addition, the 2'-OH group of the above-mentioned trisaccharide was reduced to a 2'-deoxy group and substituted 2'-O-methyl group.

Binding Sites↗

PubMed1992

Control of glycoprotein synthesis. Characterization of (1-->4)-N-acetyl-beta-D-glucosaminyltransferases acting on the alpha-D-(1-->3)- and alpha-D-(1-->6)-linked arms of N-linked oligosaccharides.

Hen oviduct membranes contain at least three N-acetyl-beta-D-glucosaminyltransferases (GlcNAc-T) that attach a beta GlcNAc residue in (1-4)-linkage to a D-Man p residue of the N-linked oligosaccharide core, i.e., (1-->4)-beta-D-GlcNAc-T III which adds a "bisecting" GlcNAc group to form the beta-D-GlcpNAc-(1-->4)-beta-D-Man p-(1-->4)-D-GlcNAc moiety; (1-->2)-beta-D-GlcNAc-T IV which adds a GlcNAc group to the (1-->3)-alpha-D-Man arm to form the beta-D-GlcpNAc-(1-->4)-[beta-D- GlcpNAc-(1-->2)]-alpha-D-Man p-(1-->3)-beta-D-Man p-(1-->4)-D-GlcpNAc component; and (1-->4)-beta-D-GlcNAc-T VI which adds a GlcNAc group to the alpha-D-Man p residue of beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpNAc- (1-->2)]-alpha-D-Man p-R to form beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpNAc-(1-->4)]-[beta-D-GlcpNAc- (1-->2)]-alpha-D-Man p-R. We now report a novel (1-->4)-beta-D-GlcNAc-T activity (GlcNAc-T VI') in hen oviduct membranes that transfers GlcNAc to beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Man p-R to form beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p-(1-->6)- beta-D-Man p-R. The structure of the enzyme product was confirmed by 1H NMR spectroscopy, FAB-mass spectrometry and methylation analysis. Previous work with GlcNAc-T IV was carried out with biantennary substrates; we now show that hen oviduct membrane GlcNAc-T IV can also transfer GlcNAc to monoantennary beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->3)-beta-D-Man p-R to form beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p- (1-->3)-beta-D-Man p-R. The findings that GlcNAc-T VI' and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl beta-D-GlcpNAc-(1-->2)-alpha-D-Man p to methyl beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p suggest that these two activities may be due to the same enzyme. The R-group of the beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Man p (or Glcp)-R substrate has an important influence on GlcNAc-T VI' enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗