A study of membrane glycoproteins from the rat brain using D-galactose-specific lectin

Immobilized D-galactose-specific lectin from Zea mais was used to purify rat brain membrane glycoproteins. The membrane glycoproteins preliminarily washed from soluble proteins were solubilized consecutively by 2% triton X-100 and 1% SDS. PAG-electrophoresis with SDS and 2-mercaptoethanol revealed 10 polypeptide bands (Mm 109, 62, 59, 54, 51, 42, 16, 14, 12.5 and 12 kDa) in the membrane fraction of glycoproteins solubilized with triton X-100. Additional solubilization with SDS revealed 3 bands (Mm 109, 62, and 54 kDa). Only 3 polypeptide bands (Mm 62, 59, 42 kDa) were identified when analogous procedure was used for purification of the rat liver glycoproteins. Horse radish peroxidase labelled D-galactose-specific lectin from Zea mais was found to bind to neuron bodies and neurites in the cerebellum. It is suggested that the identified brain-specific membrane glycoproteins may take part in the cell adhesion between neurons.     

Chromatographic separation of asparagine-linked oligosaccharides labeled with an ultravioletabsorbing compound,p-aminobenzoic acid ethyl ester

We have expanded on the suitability ofp-aminobenzoic acid ethyl ester as an ultraviolet-absorbing reagent [Wanget al., (1984) Anal Biochem 141:366–81] for the analysis of asparagine-linked oligosaccharides derived from glycoproteins. The oligosaccharides released from glycoproteins by hydrazinolysis/N-reacetylation were derivatized withp-aminobenzoic acid ethyl ester and the derivatives were purified and separated into neutral and acidic oligosaccharides on a PRE-SEP C₁₈ cartridge. The acidic oligosaccharides could be further separated into a few species by high-voltage paper electrophoresis. p-Aminobenzoic acid ethyl ester derivatives of neutral oligosaccharides were analyzed by gel permeation chromatography on Bio-Gel P-4 and HPLC on a silica-based amide column. The elution profile and the proportion of the oligosaccharides were in agreement with literature values. The overall yield of oligosaccharides from glycoproteins was approximately 70%. Fifty pmol of oligosaccharide were detectable on Bio-Gel P-4 and 4–5 pmol on HPLC.Abbreviations HPLC high performance liquid chromatography - NABEE p-aminobenzoic acid ethyl ester - FAB-MS fast-atom bombardment mass spectrometry - (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄, (GlcNAc)₅ and (GlcNAc)₆ chito-oligosaccharides containing 2,3,4,5 and 6 residues ofN-acetylglucosamine     

The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins

Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiCl extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiCl extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiCl extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiCl extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cell-wall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDa kilodalton - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Detection and immunolocalization of glycoproteins of the plasma membrane of maize sperm cells

Summary After purifying plasma membranes from isolated maize sperm cells by aqueous polymer two-phase partition, peripheral and integral proteins were solubilized from the plasma membrane with Triton X-114 and separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining revealed 10 bands (19–68 kDa) of peripheral membrane proteins and about 40 bands (12–120 kDa) of integral proteins. Peroxidase-conjugated Con A was used to detect the surface glycopeptides. It was found that Con A particularly stained 8 peripheral polypeptide bands, including 68, 66, 55, 51,48, 44, 36, and 32 kDa, and 6 integral polypeptide bands, 68, 51, 48, 44, 38, and 34 kDa. These bands differed from those of somatic samples. Staining specificity was demonstrated by the control in the presence of competing inhibitory sugar. The above result indicates the existence of mannosyl and glucosyl residues in the surface glycoproteins of maize sperm cells. The prominent peripheral 68 kDa polypeptide was further separated into 4 spots by isoelectric focusing and sodium dodecyl sulfate two-dimensional (IEF-SDS 2-D) electrophoresis, showing pI values from 5.5 to 5.8. Three prominent glycopeptides (68, 48, and 32 kDa) were localized on the plasma membrane of maize sperm cells via the fluorescein isothiocyanate (FITC) technique. About 25% of sperm cells showed an intense positive reaction in each immunological labelling. The results agree with our previous labelling of the surface of isolated viable maize sperm cells with Con A-FITC.Abbreviations FITC fluorescein isothiocyanate - Con A Canavalia ensiformis agglutinin - HRP horseradish peroxidase - RCA Ricinus communis agglutinin - WGA Triticum vulgaris agglutinin     

Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins

Summary The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3° and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures.The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous.The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa. These data suggest that the polypeptides were formed by cleavage of glutamic acid residues present at regular intervals in the chains of all three mucin glycoproteins. These large immunoreactive peptides were formed by the removal of smaller peptides from the carboxyl terminal end of the deglycosylated mucin glycoprotein chains. Taken collectively, these findings indicate that the polypeptide chains in these mucin glycoproteins are very similar in subunit structure and that there is a high degree of homology between their polypeptide chains.     

Protein profiling in F<Subscript>1</Subscript> and F<Subscript>2</Subscript> generations of two tomato genotypes differing in ripening time

Pericarp polypeptide profiles were analyzed at three ripening stages in the F₁ hybrid and the F₂ population from the cross between the accessions: LA1385 (Lycopersicon esculentum var. cerasiforme) and 804627 (L. esculentum, a homozygous genotype for the nor mutant). Six polymorphic polypeptides were observed in LA1385, while no polymorphic polypeptides among ripening stages was observed in 804627. On the other hand, some polypeptides in the F₁ hybrid were not observed in the parents whereas others were present in both parental genotypes and were unnoticeable in the hybrid genotype. From a cluster analysis on the protein profiles of the F₂ population, the differential expression of proteins allowed to distinguish mature green (MG) stage from the others two stages, while for breaker stage (BR) and red ripe stage, the genetic background was more important in forming groups. The differential expression of proteins could be associated with fruit morphology traits such as a 72 kDa polypeptide present in MG stage with fruit diameter, height and mass and a 47 kDa polypeptide found in BR with fruit shelf life.     

Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae.

Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.     

Effect of Hypoxia on Endothelial Cell Surface Glycoprotein Expression: Modulation of Glycoprotein IIIa and Other Specific Surface Glycoproteins

Exposure to hypoxia alters many aspects of endothelial cell metabolism and function; however, changes in surface glycoconjugates under these conditions have not been extensively evaluated. In the current studies, we examined surface glycoproteins of cultured bovine aortic (BAEC) and pulmonary arterial (BPAEC) endothelial cells under standard culture conditions (21% oxygen) and following exposure to hypoxia (0% oxygen) for varying time periods (30 min to 18 h) using a system of biotinylation, lectin binding (concanavalin A, Con A; Griffonia simplicifolia , GSA; Arachis hypogaea, PNA; Ricinus communis, RCA; or Triticum vulgaris, WGA), subsequent strep-avidin binding, and staining. Using these methods, we identified differences in lectin binding between the two cell types cultured in 21% oxygen with all lectins except PNA. With exposure to 0% oxygen, there was no change in lectin binding to most surface glycoproteins. Several surface glycoproteins, including glycoprotein IIIa on both cell types, demonstrated a time-dependent decrease in lectin binding; in addition, there was an increase in lectin binding to a few specific surface glycoproteins on each cell type within 30-60 min of exposure to 0% oxygen. These changes in specific surface glycoproteins were confirmed in both cell types by ¹²⁵I labeling. Increased lectin binding was observed for Con A binding BAEC glycoproteins at molecular weight (MW) 116, 130, and 205 kDa, GSA binding BAEC glycoproteins at MW 120 and 205 kDa, and RCA binding BPAEC glycoproteins at MW 140 and 205 kDa. Increased binding of WGA or PNA was not observed during exposure to hypoxia. The specificity of lectin binding was further confirmed by competitive inhibition with the appropriate sugar. These studies demonstrate that there are baseline differences between BAEC and BPAEC cell surface glycoproteins and that exposure to hypoxia is associated with little change in lectin binding to most surface glycoproteins. There is, however, increased surface expression of a few glycoproteins that differ depending of the origin of the endothelial cell. Although the mechanism of this increase in lectin binding is not yet clear, subsequent studies suggested that it is due to increased availability of select carbohydrate moieties. The time course of these alterations suggests a possible role in the endothelial cell response to decreases in ambient oxygen tension.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

Cloning and Characterization of Two Novel Crystal Protein Genes, <Emphasis Type="Italic">cry54Aa1</Emphasis> and <Emphasis Type="Italic">cry30Fa1</Emphasis>, from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain BtMC28

Bacillus thuringiensis strain BtMC28 was isolated from the soil sample in China. Two novel crystal protein genes were found by using the PCR-RFLP method. Moreover, the full-length sequences of two novel genes were obtained by a single oligonucleotide nested (SON)-PCR upstream and downstream strategy. Sequence analysis revealed that one gene encoded a polypeptide of 673 amino acid residues with a molecular mass of 76.3 kDa, 38% identical to Cry10Aa, and the other encoded a polypeptide of 687 amino acid residues with a molecular mass of 77.1 kDa, 74% identical to Cry30Aa. These two novel crystal protein genes were designated as cry54Aa1 and cry30Fa1 by Bt Insecticidal Crystal Proteins Nomenclature Committee, respectively. The Cry54Aa1 and Cry30Fa1 proteins retained five conserved regions commonly found in the existing Cry proteins. Cry54Aa1 protein exhibited insecticidal activities against Laphygma exigua (Lepidoptera), Helicoverpa armigera (Lepidoptera), and Aedes aegypti (Diptera) when its encoding gene was expressed in an Escherichia coli host strain. The authors, Furong Tan and Jun Zhu contributed equally to this work.     

A journey to the world of glycobiology

Finding of the deletion phenomenon of certain oligosaccharides in human milk and its correlation to the blood types of the donors opened a way to elucidate the biochemical basis of blood types in man. This success led to the idea of establishing reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins. N-Linked sugar chains were first released quantitatively as oligosaccharides by enzymatic and chemical means, and labelled by reduction with NaB³H₄. After fractionation, structures of the radioactive oligosaccharides were determined by a series of methods developed for the studies of milk oligosaccharides. By using such techniques, structural rules hidden in the N-linked sugar chains, and organ- and species-specific N-glycosylation of glycoproteins, which afforded a firm basis to the development of glycobiology, were elucidated. Finding of galactose deficiency in the N-linked sugar chains of serum lgG from patients with rheumatoid arthritis, and malignant alteration of N-glycosylation in various tumors opened a new research world called glycopathology.However, recent studies revealed that several structural exceptions occur in the sugar chains of particular glycoproteins. Finding of the occurrence of the Gal1-4Fuc1- group linked at the C-6 position of the proximal N-acetylglucosamine residue of the hybrid type sugar chains of octopus rhodopsin is one of such examples. This finding indicated that the fucosyl residue of the fucosylated trimannosyl core should no more be considered as a stop signal as has long been believed. Furthermore, recent studies on dystroglycan revealed that the sugar chains, which do not fall into the current classification of N- and O-linked sugar chains, are essential for the expression of the functional role of this glycoprotein.It was found that expression of many glycoproteins is altered by aging. Among the alterations of the glycoprotein patterns found in the brain nervous system, the most prominent evidence was found in P₀. This protein is produced in non-glycosylated form in the spinal cord of young mammals. However, it starts to be N-glycosylated in the spinal cord of aged animals.These evidences indicate that various unusual sugar chains occur as minor components in mammals, and play important roles in particular tissues.     

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: Sequence similarity to PSI-K

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.     

Sialylation of N-Carbohydrate Chains of Glycoproteins with Trans-Sialidase from Trypanosoma cruzi

2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. ruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for 2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.     

Cloning and characterization of a cDNA encoding a Rab1-like small GTP-binding protein from Petunia hybrida

The cloning of small GTP-binding proteins from Petunia hybrida was performed using a PCR-based strategy. Degenerate primers were designed from the DTAGQE and FMETSA consensus sequences. Three different cDNAs were amplified. The deduced polypeptide sequences PhPCRGP1 and PhPCRGP2 were homologous to RB11_HUMAN and PhPCRGP3 to RAB1A_HUMAN. Using PhPCRGP3 as a probe, 8 identical clones were selected from a Petunia leaf cDNA library. They all encode the same 22.5 kDa polypeptide, PhRAB1, able to bind GTP in vitro and 72% identical to RAB1A_HUMAN. Hybridizable mRNAs encoding PhRAB1 accumulated preferentially in opened flowers.     

Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine.

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.     

N-Glycosylation of membrane glycoproteins in retinol-deficient rat liver

The effect of vitamin A deficiency onN-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in proteinN-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled withd-[2-³H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc₁Man₉GlcNAc₂, Man₉GlcNAc₂, Man₈GlcNAc₂ and Man₇GlcNAc₂, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number ofN-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis ofN-linked oligosaccharides of glycoproteins must be questioned.Abbreviations DolP Dolichyl phosphate - DolPP dolichyl pyrophosphoryl - RetPMan retinyl phosphate mannose - DPP IV dipeptidyl peptidase IV (EC 3.4.14.5) - endo H endo--N-acetylglucosaminidase H (EC 3.2.1.96) - endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Antigenic and structural analysis of Treponema denticola

Polypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determined by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product of a larger, heat-modifiable polypeptide. Based on the results of SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a 'rough' lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology of the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath and core structure and polypeptide composition characteristic of that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure of antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detailed molecular analysis of the ultrastructure and antigenicity of T. denticola.     

Specificity of action on mosquito larvae of Bacillus thuringiensis israelensis toxins encoded by two different genes

Summary A 135 kDa protein gene and two open reading frames (ORF1 and ORF2) have been cloned from a large plasmid of Bacillus thuringiensis israelensis (Bourgouin et al. 1986). The Escherichia coli recombinant clones containing these genes were highly toxic to larvae of Aedes aegypti, Anopheles stephensi and Culex pipiens. From subcloning experiments it was deduced that the 135 kDa polypeptide alone was responsible for the toxic activity on both A. aegypti and An. stephensi larvae. In contrast, the presence of two polypeptides, the 135 kDa protein and the ORF1 product was required for toxicity to C. pipiens larvae. The minimal toxic fragment of the 135 kDa polypeptide has been delineated. The results indicate that a polypeptide of about 65 kDa, corresponding to an amino-terminal part of the 135 kDa protein is sufficient for toxicity. Sequence comparisons indicate that the ORF1 product may correspond to an N-terminal part of a rearranged 130 kDa protein.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

The third chains of living organisms--a trail of glycobiology that started from the third floor of building 4 in NIH

Application of a finger-printing method to the analysis of human milk oligosaccharides led to the finding that several oligosaccharides were missing in the milk of non-secretor or Lewis-negative individuals. This finding helped us in opening the door of elucidating the enzymatic basis of blood types in human. Based on these successful studies, a strategy to establish reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins was devised. It was to contrive enzymatic and chemical means to release quantitatively the N-linked sugar chains as oligosaccharides, and finger-print them by using appropriate methods to demonstrate the sugar pattern of a glycoprotein. These methods enabled us to determine that the N-linked sugar chains of glycoproteins can be classified into three subgroups: high mannose-type, complex-type, and hybrid-type. By comparative studies of the sugar patterns of a glycoprotein produced by different organs and different animals, occurrences of organ- and species-specific glycosylation were found in many glycoproteins. By comparative studies of the glycosylation patterns of the subunits constructing human chorionic gonadotropin and other glycoproteins, occurrence of site-directed N-glycosylation was also found, indicating that the processing and maturation of the N-linked sugar chains of a glycoprotein might be controlled by the structure of polypeptide moiety. Furthermore, these methods enabled us to elucidate the structural alteration of the sugar chains of a glycoprotein induced by diseased state of the producing cells, such as rheumatoid arthritis and malignancy. Recent studies of glycoproteins in the brain-nervous system through aging revealed that N-glycosylation of P(0) in the rat spinal cord is induced by aging. Therefore, glycobiology is expanding tremendously into fields such as pathological and gerontological research.