scholarly journals Characterization of the oligosaccharide component of arylsulfatase B from rat liver.

1997 ◽  
Vol 44 (2) ◽  
pp. 181-190 ◽  
Author(s):  
M Przybyło ◽  
A Lityńska
Keyword(s):  
Rat Liver ◽  
Gel Permeation Chromatography ◽  
Complex Type ◽  
Relative Contribution ◽  
Chain Analysis ◽  
Terminal Galactose ◽  
Glycan Chain ◽  
Arylsulfatase B ◽  
High Mannose

A glycan chain analysis of the total oligosaccharide pool derived from rat liver arylsulfatase B was carried out by. P4 Gel Permeation Chromatography and sequential exoglycosidase digestion. It was found that 71% of rat liver arylsulfatase B oligosaccharides were sialylated. The relative contribution of particular structures in the total glycan pool was as follows: sialylated biantennary complex type glycans with terminal galactose--65%, high-mannose type glycans--15%, biantennary complex type glycans with core fucose and terminal N-acetylglucosamine--5%, O-linked oligosaccharides--3.5%.

scholarly journals Preliminary characterization of the oligosaccharide component of arylsulfatase B from human placenta.

1995 ◽  
Vol 42 (1) ◽  
pp. 45-50
Author(s):  
P Laidler ◽  
M Gałka-Walczak ◽  
A Lityńska
Keyword(s):  
Sialic Acid ◽  
Human Placenta ◽  
Carbohydrate Component ◽  
Lectin Affinity Chromatography ◽  
Complex Type ◽  
Total Carbohydrate ◽  
Chain Analysis ◽  
Glycan Chain ◽  
Arylsulfatase B

Isoelectric focusing of homogenous arylsulfatase B from human placenta pointed to the presence of enzymatically active and inactive forms of high pI (pH 9-8) and of lower pI (pH 6.5-5.5). Glycan chain analysis performed with the use of a Glycan Differentiation Kit showed that basic forms of arylsulfatase B from human placenta contained mostly high mannose/hybrid type glycans, with 6-O-L-fucose bound to the innermost N-acetylglucosamine residue, whereas acidic forms of the enzyme contained complex type glycans containing fucose and sialic acid. However, the latter forms constitute a small percentage of the total carbohydrate component. Lectin affinity chromatography of the native enzyme confirmed the presence of a core fucose and a sialic acid.

scholarly journals Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits

1986 ◽  
Vol 239 (3) ◽  
pp. 679-683 ◽  
Author(s):  
A McElduff ◽  
A Watkinson ◽  
J A Hedo ◽  
P Gorden
Keyword(s):  
Insulin Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Beta Subunits ◽  
Relative Distribution ◽  
Single Chain ◽  
Complex Type ◽  
Mannose Residue ◽  
Predominant Species ◽  
High Mannose

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

scholarly journals Partial characterization of the oligosaccharides of mouse thymocyte Thy-1 glycoprotein

1984 ◽  
Vol 221 (2) ◽  
pp. 379-392 ◽  
Author(s):  
S R Carlsson ◽  
T Stigbrand
Keyword(s):  
Gel Filtration ◽  
Glycosylation Site ◽  
The Other ◽  
Complex Type ◽  
Mouse Thymocyte ◽  
Lentil Lectin ◽  
High Mannose ◽  
Sepharose 4B ◽  
Oligosaccharide Structures

Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary ‘complex-type’ with terminal fucose; IIA, biantennary ‘complex-type’ without fucose; IIB, biantennary ‘complex-type’ with fucose; III, a mixture of ‘high-mannose’ chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of ‘high-mannose’ type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with ‘complex-type’ chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three ‘complex-type’ chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.

scholarly journals X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

2017 ◽  
Vol 73 (10) ◽  
pp. 822-828 ◽  
Author(s):  
Harry B. Gristick ◽  
Haoqing Wang ◽  
Pamela J. Bjorkman
Keyword(s):  
Crystal Structures ◽  
Biochemical Characterization ◽  
Complex Type ◽  
Cryo Electron Microscopy ◽  
High Mannose ◽  
Complex Glycans ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Trimeric Envelope

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.

Characterization by nuclear magnetic resonance of the concanavalin A binding oligosaccharide on the βb chain of placental β-hexosaminidase B: lectin binding to the separated polypeptide chains of hexosaminidases A and B

1985 ◽  
Vol 63 (7) ◽  
pp. 723-729 ◽  
Author(s):  
Brian F. O'dowd ◽  
Don Mahuran ◽  
Dale Cumming ◽  
J. Alexander Lowden
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Concanavalin A ◽  
Lectin Binding ◽  
Proton Nuclear Magnetic Resonance ◽  
Complex Type ◽  
High Mannose ◽  
Polypeptide Chains ◽  
Nuclear Magnetic

The type and distribution of the oligosaccharides on each polypeptide of human placental β-hexosaminidases A (α(βaβb)) and B (2((βaβb)) were examined. The denatured polypeptides were separated by isoelectric focussing in a polyacrylamide slab gel and each gel was then overlaid with 125I-labelled lectins. The study indicated that the βa chain contains negligible carbohydrate, the βb chain contains both the high-mannose and a complex type oligosaccharide, and the α chain contains predominantly high-mannose or hybrid type moieties. Two asparagine-linked high-mannose type oligosaccharides present on the βb polypeptide of β-hexosaminidase B were isolated by concanavalin A chromatography and by reverse-phase high pressure liquid chromatography. Proton nuclear magnetic resonance characterization of the oligosaccharides revealed an equimolar glycan mixture of the high-mannose type structure Man5 and Man6.

Characterization of the oligosaccharides of liver Z variant α1-antitrypsin

1980 ◽  
Vol 58 (8) ◽  
pp. 644-648 ◽  
Author(s):  
Albert Hercz ◽  
Noam Harpaz
Keyword(s):  
Endoplasmic Reticulum ◽  
Sialic Acid ◽  
Sindbis Virus ◽  
Smooth Endoplasmic Reticulum ◽  
Gel Permeation Chromatography ◽  
New Product ◽  
Complex Type ◽  
Homogeneous Product ◽  
Normal Individuals

The plasma α1-antitrypsin (AT) of normal individuals has been reported to have four oligosaccharides per molecule; these oligosaccharides are all of the complex type, containing only three D-mannose (Man) residues per oligosaccharide as well as several residues of galactose, sialic acid, and N-acetyl-D-glucosamine (GlcNAc) (Chan, S. K., Rees, D. C., Li, S.-C. &Li, Y.-T. (1976) J. Biol. Chem. 251, 471–476; Hodges, L. C., Laine, R. &Chan, S. K. (1979) J. Biol. Chem. 254, 8208–8212). Analysis of the carbohydrates of purified liver Z variant AT was consistent with the presence of three moles of Man-rich oligosaccharide per mole protein with an average of seven Man and two GlcNAc residues per oligosaccharide and a complete absence of galactose and sialic acid (Hercz, A., Katona, E., Cutz, E., Wilson, J. R., &Barton, M. (1978) Science 201, 1229–1232). We have now carried out a more definitive structural analysis of the oligosaccharides of liver Z variant. Glycopeptides were prepared from purified liver Z variant by digestion with pronase. Treatment of a sample of the glycopeptides with endo-β-N-acetylglycosaminidase CII (endo CII) resulted in the formation of three oligosaccharides. These were identified by gel permeation chromatography on a Biogel P-4 column, calibrated with a series of ovalbumin and Sindbis virus oligosaccharides, as GlcNAcMan5, GlcNAcMan6, and GlcNAcMan7. Treatment of a sample of the glycopeptides with endo-β-N-acetylglycosaminidase D gave rise only to a single oligosaccharide product, GlcNAcMan5. Upon incubation of the glycopeptides with UDP[U-14C]GlcNAc in the presence of purified bovine colostrum UDPGlcNAc:α-D-mannoside α-2-N-acetylglucosaminyltransferase I, AsnGlcNAc2Man5 quantitatively disappeared and a new product consistent with the structure AsnGlcNAc2Man5[14C]GlcNAc was formed. Incubation of the oligosaccharides, formed with endo CII, with α-mannosidase gave rise to a 90% homogeneous product with the probable structure of Manβ1 → 4GlcNAc. These findings suggest that the precursors of the complex oligosaccharides of the secretory glycoprotein, plasma AT, are Man-rich oligosaccharides. It appears also that the glycosidases which process the precursor to the AsnGlcNAc2Man5 stage are present and active in the rough endoplasmic reticulum of Pi (protease inhibitor) ZZ individuals; however, for an as yet undetermined reason, most of the Z variant AT appears unable to migrate at a normal rate to the smooth endoplasmic reticulum and Golgi apparatus for eventual secretion from the liver.

Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽  
2000 ◽  
pp. 325-335 ◽  
Author(s):  
A Calvo ◽  
LM Pastor ◽  
S Bonet ◽  
E Pinart ◽  
M Ventura
Keyword(s):  
Ricinus Communis ◽  
Lectin Histochemistry ◽  
Apical Part ◽  
Seminiferous Tubules ◽  
In Situ Characterization ◽  
Intense Staining ◽  
Terminal Galactose ◽  
Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

scholarly journals Growth, lifetime, directional movement and myosin-dependent motility of mutant keratin granules in cultured cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. M. Lehmann ◽  
R. E. Leube ◽  
R. Windoffer
Keyword(s):  
Digital Image Analysis ◽  
Cultured Cells ◽  
Epidermolysis Bullosa Simplex ◽  
Continuous Growth ◽  
Relative Contribution ◽  
Complete Life Cycle ◽  
Directional Movement ◽  
Multiple Characteristics ◽  
Cell Centre

AbstractIntermediate filament polypeptides (IFPs) are prominent components of cytoplasmic aggregates, which are pathognomonic for multiple diseases. Recent observations in cultured cells suggest that they are dynamic and subject to regulated turnover. The emerging concept is that multiple factors contribute to motility and turnover of IFP-containing aggregates. To understand their relative contribution, quantitative tools are needed. The current study addresses this need using epithelial cells producing mutant keratin IFPs that have been identified as the cause of the hereditary blister-forming skin disease epidermolysis bullosa simplex. Digital image analysis of individual granules allowed mapping of their complete life cycle, with information on multiple characteristics at any given time-point. The deduced signet features revealed rapid granule fusion and directed transport from the periphery towards the cell centre, and a limited, ~ 30 min lifetime with a slow, continuous growth phase followed by fast disassembly. As paradigmatic proof-of-principle, we demonstrate that inhibition of myosin II selectively reduces granule movement, linking keratin granule motility to retrograde cortical acto-myosin flow. The newly developed methods and established parameters will help in the characterization of known and the identification of novel regulators of IFP-containing aggregates.

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