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H Paulsen

Publications and source records attributed to H Paulsen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Decreasing the chlorophyll a/b ratio in reconstituted LHCII: structural and functional consequences.

Trimeric (bT) and monomeric (bM) light-harvesting complex II (LHCII) with a chlorophyll a/b ratio of 0.03 were reconstituted from the apoprotein overexpressed in Escherichia coli. Chlorophyll/xanthophyll and chlorophyll/protein ratios of bT complexes and 'native' LHCII are rather similar, namely, 0.28 vs 0. 27 and 10.5 +/- 1.5 vs 12, respectively, indicating the replacement of most chlorophyll a molecules with chlorophyll b, leaving one chlorophyll a per trimeric complex. The LD spectrum of the bT complexes strongly suggests that the chlorophyll b molecules adopt orientations similar to those of the chlorophylls a that they replace. The circular dichroism (CD) spectra of bM and bT complexes indicate structural arrangements resembling those of 'native' LHCII. Thermolysin digestion patterns demonstrate that bT complexes are folded and organized like 'native' trimeric LHCII. Surprisingly, in the bT complexes at 77 K, half of the excitations that are created on either chlorophyll b or xanthophyll are transferred to chlorophyll a. No or very limited triplet transfer from chlorophyll b to xanthophyll appears to take place. However, the efficiency of triplet transfer from chlorophyll a to xanthophyll is close to 100%, even higher than in 'native' LHCII at 77 K. It is concluded from the triplet-minus-singlet and CD results that the single chlorophyll a molecule that on the average is present in each bT complex binds preferably next to a xanthophyll molecule at the interface between the monomers.

Absorption↗
PubMed1999

Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-Acetylgalactosamine:Peptide N-acetylgalactosaminyltransferases. site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions.

In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitro addition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcalpha or Galbeta1-3GalNAcalpha at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position -6 (Thr-10) or -10 (Ser-6) and is inhibited by disaccharide at position -11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.

Acetylgalactosamine↗
PubMed1999

Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry.

Sequencing of eight O-glycosylated peptides by nanoESI-QTOF-MS/MS was carried out to provide a sensitive general characterization method for determination of glycosylation site(s) and of the type of the attached carbohydrate moiety in a single experiment. The glycopeptide structures were chosen to demonstrate the feasibility of this sensitive and accurate approach, where isobaric peptide structures either (i) with the same number of attachment sites in different position in the peptide backbone, and (ii) with the same number of sugar moieties distributed on different attachment sites in the peptide backbone, can be clearly distinguished. Beside the B-type carbohydrate sequence ions of high abundance, it is possible to register diagnostic b- and y-type glycosylated peptide ions of lower abundance due to high dynamic range of the QTOF analyser. The applicability of this approach for detailed analysis of highly clustered O-glycan structures as found in biological mucin samples is discussed.

Glycosylation↗
PubMed1999

'Wave-type' structure of a synthetic hexaglycosylated decapeptide: a part of the extracellular domain of human glycophorin A.

The three-dimensional structure of a glycopeptide, His-Thr*-Ser*-Thr*-Ser*-Ser*-Ser*-Val-Thr-Lys, with 2-acetamido-2-deoxy-alpha-D-galactose (GalNAc) residues linked to six adjacent amino acids from Thr-10 to Ser-15, was studied by NMR spectroscopy and molecular dynamics (MD) simulations. The hexaglycosylated decapeptide is part of the extracellular domain of human glycophorin A and shows an extended structure of the peptide backbone due to O-glycosylation. Furthermore, each GalNAc residue exhibits one and only one NOE contact from the NHAc proton to the backbone amide proton of the amino acid that the sugar is directly bound to. This indicates a strong preference for the orientation of all GalNAc residues towards the N-terminus. NOE build-up curves were used to determine 42 inter-proton distances that, in connection with phi angles of the peptide backbone obtained from 3J-coupling constants, resulted in constraints for a MD simulation in water. The NMR data and the MD simulations show a preference for an extended backbone structure. The GalNAc residues are located alternatingly on opposite sides of the backbone and reduce the flexibility of the peptide backbone. The conformation of the molecule is relatively rigid and shows a 'wave-type' 3D structure of the peptide backbone within the glycosylation cluster. This new structural element is also supported by the unusual CD spectrum of the glycopeptide.

Acetylgalactosamine↗
PubMed1999

Expression of a higher plant light-harvesting chlorophyll a/b-binding protein in Synechocystis sp. PCC 6803.

A chimeric lhcb gene, coding for Lhcb, a higher plant chlorophyll a/b-binding light-harvesting complex of photosystem II (LHCII), was constructed using the Synechocystis sp. PCC 6803 psbA3 promoter and a modified lhcb gene from pea. This construct drives synthesis of full-length, mature Lhcb under the control of the strong psbA3 promoter that usually drives expression of the D1 protein of photosystem II. This chimeric gene was transformed into a photosystem I-less/chlL(-) Synechocystis sp. PCC 6803 strain that is unable to synthesize chlorophyll in darkness. In the resulting strain, a high level of lhcb transcript was detected and transcript accumulation was enhanced by addition of exogenous Zn-chlorophyllide b. The chimeric lhcb gene was translated to produce full-length Lhcb as demonstrated by pulse-labeling: a new radioactively labeled band of a size corresponding to full-length Lhcb was visible on autoradiograms. Using Triton X-114 phase fractionation, this labeled protein band was found to partition to the phase containing integral membrane proteins, indicating that the pulse-labeled Lhcb is readily integrated into the membrane. However, Lhcb was rapidly degraded and did not accumulate in thylakoid membranes to levels that were detectable other than by pulse labeling. Upon immunological detection with LHCII antibodies, a small protein (approximately 8 kDa) was found specifically in the lhcb-containing mutant. We interpret this protein to be a degradation product of the full-length Lhcb. This fragment was stabilized by supplementing cells with xanthophylls, which incorporated into thylakoid membranes only in the mutant carrying lhcb. The lutein/chlorophyll ratio of thylakoids of this mutant was about 1 : 10. These results indicate that in this cyanobacterial system Lhcb is synthesized, integrated into the membrane, and then degraded to a approximately 8 kDa fragment that is stabilized by pigment binding and does not require the presence of chlorophyll b.

Chlorophyll↗
PubMed1998

Truncated recombinant light harvesting complex II proteins are substrates for a protein kinase associated with photosystem II core complexes.

Previous studies directed towards understanding phosphorylation of the chlorophyll alb binding proteins comprising light harvesting complex II (LHC II) have concentrated on a single phosphorylation site located close to the N-terminus of the mature proteins. Here we show that a series of recombinant pea Lhcb1 proteins, each missing an N-terminal segment including this site, are nevertheless phosphorylated by a protein kinase associated with a photosystem II core complex preparation. An Lhch1 protein missing the first 58 amino acid residues is not, however, phosphorylated. The results demonstrate that the LHC II proteins are phosphorylated at one or more sites, the implications of which are discussed.

Amino Acid Sequence↗
PubMed1998

Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation.

The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.

Antibodies, Monoclonal↗
PubMed1998

Pigment-binding properties of the recombinant photosystem II subunit CP26 reconstituted in vitro.

CP26 is the most recently described antenna protein in higher plants which has been reported to be involved in xanthophyll-dependent regulation of the light-harvesting function but is largely unknown due to the difficulties of purification. In this study we have overexpressed in Escherichia coli the Lhcb5 gene product and reconstituted CP26 in vitro by refolding the recombinant protein in the presence of chlorophyll a, chlorophyll b and xanthophylls. The resulting pigment-protein complex is stable enough to be isolated by partially denaturing gel electrophoresis. Reconstitution and isolation conditions for CP26 are similar to those used for other chlorophyll a/b complexes like the major light-harvesting complex of photosystem II (LHCII) and CP29; however, CP26 differs with regard to its lower specificity in carotenoid binding. Most significantly, rather stable recombinant CP26 can be reconstituted containing violaxanthin as the only carotenoid. This enhanced plasticity with respect to carotenoid binding is consistent with CP26 being the major binding protein of violaxanthin involved in the xanthophyll cycle. The availability of recombinant CP26 opens the way to a better characterisation of this pigment-protein complex with regard to its biochemistry and its physiological functions.

Apoproteins↗
PubMed1998

Synthesis of an N-glucoasparagine analog as a building block for a V3-loop glycopeptide from gp120 of HIV-I.

The preparative synthesis of a new N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine mimetic 1, starting from 2-amino-1,5-anhydro-2-deoxy-glucitol hydrochloride and Z-Asp-(OH)-OBn is described. This glycosyl-amino acid unit 1 is expected to show higher stabilities towards in vivo conditions. Further, the use of 1 as building block for the synthesis of modified glycopeptides using solid phase support is reported. The glycopeptide Ac-SXNTRKSIHIGPGRAF-NH2 having sugar-modified Asn2 mimics parts of the V3-loop structure containing the principle neutralizing determinant (PND) of HIV-1 and the naturally conserved glycosylation site within the V3 loop.

Acetylglucosamine↗
PubMed1997

Light-harvesting chlorophyll a/b-binding protein stably inserts into etioplast membranes supplemented with Zn-pheophytin a/b.

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Chlorophyll↗
PubMed1997

Synthesis and characterisation of highly glycosylated glycopeptides with Tn-antigenic structures corresponding to human glycophorin AN.

Two highly glycosylated O-glycopeptides corresponding to human glycophorin AN with Tn-antigenic structures were synthesised. The first glycopeptide has two glycosylated clusters with three and six adjacent 2-acetamido-2-deoxy-D-galactose (GalNAc) glycosylation sites and represents the N-terminal octadecapeptide from Leu-1 to Lys-18. The second glycopeptide, a decapeptide from His-9 to Lys-18, contains as a compact cluster six adjacent GalNAc glycosylation sites. The solid phase synthesis was realised by using the carbohydrate-containing building blocks Fmoc-Ser(Ac3GalN3)-Pfp and Fmoc-Thr(Ac3GalN3)-Pfp. The synthesised substances were characterised by NMR spectroscopic techniques. The main techniques used were homonuclear TOCSY and NOESY as well as HMQC and HMBC for 13C,1H correlations.

Amino Acid Sequence↗
PubMed1997

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Binding Sites↗
PubMed1996

The C-terminal domain of light-harvesting chlorophyll-a/b-binding protein is involved in the stabilisation of trimeric light-harvesting complex.

Light-harvesting chlorophyll a/b-binding protein (LHCP) can be reconstituted with pigments in detergent solution to yield stable monomeric light-harvesting chlorophyll a/b complex (LHCII). This reconstitution is not significantly affected when up to ten amino acids are deleted on the C-terminus of LHCP or when a tryptophan, which is 11 positions from the C terminus (W222), is exchanged with other amino acids [Paulsen, H. & Kuttkat, A. (1993) Photochem. Photobiol. 57, 139-142]. Here we show that the exchange of W222 with histidine or glycine completely abolishes the ability of the protein to assemble into trimeric LHCII, either upon reconstitution of monomeric complexes in detergent solution or upon insertion into isolated thylakoids. It is concluded that part of the hydrophilic domain on the C-terminus of LHCP, although not essential for the formation of stable monomeric LHCII, is involved in trimer formation. The different degree to which various amino acids in place of W222 affect trainer formation suggests that a hydrophobic amino acid is needed in this position.

Amino Acid Sequence↗
PubMed1996

Assembly of light-harvesting chlorophyll a/b complex in vitro. Time-resolved fluorescence measurements.

The assembly kinetics of a pigment-binding membrane protein, the light- harvesting chlorophyll a/b complex of green plants, have been determined in vitro. Time-resolved fluorescence spectroscopy, with millisecond time resolution, has been used to monitor changes in both protein and chlorophyll (Chl) fluorescence, as well as in energy transfer from Chl b to Chl a, during complex assembly. Three reaction steps could be resolved after rapid, stopped-flow, mixing of the apoprotein (light-harvesting Chl a/b protein, LHCP) and pigments, solubilized in sodium dodecyl sulfate (SDS) and octyl glucoside (OG), respectively. A fast step in the range of 10 ms was detected regardless of whether the reaction mixture contained pigments or proteins, or both, and is interpreted as being connected with the formation of mixed SDS and OG detergent micelles. Two further steps were resolved: one with a time constant of about 1 min and another, slow step with a time constant of several minutes. Both of these were dependent on the presence of protein, Chls, and xanthophylls. Most, if not all, of the energy transfer from Chl b to Chl a was established during the slow step, indicating the the juxtaposition of these pigments, either by a structural rearrangement of the complex of by additional pigment binding, is the final stage in LHCII assembly in vitro.

Apoproteins↗
PubMed1996

Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase using synthetic glycopeptide substrates.

The factors determining glycosylation of mucin type glycoproteins are not well understood. In the present work, we investigated the role of the peptide moiety and of the presence of O-glycan chains on O-glycosylation by UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyl-transferase (ppGalNAc-T). We used purified ppGalNAc-T from bovine colostrum and a series of synthetic glycopeptide and peptide substrates most of which contained sequences derived from the tandem repeat region of MUC2 mucin. The rate of incorporation of GalNAc into Thr was significantly greater than toward Ser residues. The presence of one or two GalNAc-Thr moieties in the substrate significantly reduced enzyme activity, and this effect was more pronounced when the disaccharide Gal beta 1-3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Gal beta 1-3GalNAc-Thr occurs at a slower rate than primary glycosylation of carbohydrate-free peptide. Analysis of products by HPLC showed that the enzyme was selective in glycosylating peptides or glycopeptides with the PTTTPIST sequence in that the preferred primary glycosylation site was the third Thr from the amino-terminal end; secondary glycosylation depended on the site of the primary glycosylation. Negatively but not positively charged amino acids on the carboxy-terminal side of the putative secondary glycosylation site resulted in high activity suggesting charge-charge interactions of substrates with the enzyme. These studies indicate that O-glycosylation by bovine colostrum ppGalNAc-T is a selective process dependent on both the amino acid sequence and prior glycosylation of peptide substrates.

Animals↗
PubMed1996

Glycopeptide mimics of mammalian Man9GlcNAc2. Ligand binding to mannan-binding proteins (MBPs).

A novel and simple approach for rational design of oligosaccharide mimics has been developed. Mammalian high-mannose triantennary structure Man9GlcNac2 has been subjected to molecular modelling using the NMR data available on structural fragments of the oligosaccharide. The analysis indicated four different low energy conformations, and the spatial arrangement of terminal disaccharides of the oligosaccharide antennae were stimulated with glycopeptides carrying disaccharides by applying weak constraints between the saccharide parts in MD-simulations on a large array of tri- to octaglycopeptides. The five glycopeptides exhibiting the best fit with the four minimum energy confirmations of the oligosaccharide were synthesized by solid phase glycopeptide assembly using glycosylated fluoren-9-ylmethyloxycarbonyl-amino acid-O-pentafluorophenyl esters as building blocks. The glycan was acyl protected alpha-D-Man-(1-->2)-alpha-D-Man and Ser, Thr and Hyp were the glycosylated amino acids. The deprotected and purified glycopeptides were subjected to NMR analysis for characterization, and in order to investigate the cis-trans isomerism of the carbimide bonds to Hyp. The glycopeptides were tested for their ability to inhibit binding of mannan-binding protein to mannan from Saccharomyces cerevisiae. They were found to be weak inhibitors showing no indication of multivalent interaction with the mannan-binding protein.

Carbohydrate Conformation↗
PubMed1995

Synthesis of pentasaccharide analogues of the N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V using tetrasaccharide precursors and recombinant beta-(1-->2)-N-acetylglucosaminyltransferase II.

Recombinant human UDP-GlcNAc: alpha-Man-(1-->6)R beta-(1-->2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143, GlcNAc-T II) was produced in the Sf9 insect cell/baculovirus expression system as a fusion protein with a (His)6 tag and partially purified by affinity chromatography on a metal chelating column. The partially purified enzyme was used to catalyze the transfer of GlcNAc from UDP-GlcNAc to R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3))beta-Man-O-octyl to form beta-GlcNAc(1-->2)R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha- Man(1-->3))beta-Man-O-octyl where there is either no modification of the alpha-Man(1-->6) residue (7), or where R is 3-deoxy (8), 4-deoxy (9) or 6-deoxy (10). The yields ranged from 64-80%. Products were characterized by 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 7-10 are pentasaccharide analogues of the biantennary N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V.

Carbohydrate Conformation↗