The efficiency of N-linked glycosylation of bovine DNase I depends on the Asn-Xaa-Ser/Thr sequence and the tissue of origin

2001 ◽  
Vol 355 (1) ◽  
pp. 245-248 ◽  
Author(s):  
Atsushi NISHIKAWA ◽  
Sumi MIZUNO
Keyword(s):  
Submaxillary Gland ◽  
Dnase I ◽  
The State ◽  
Sugar Chain ◽  
Glycosylation Sites ◽  
Tissue Of Origin ◽  
Sugar Chains

Bovine DNase I contains two potential N-linked glycosylation sites with the sequences Asn18-Ala-Thr and Asn106-Asp-Ser. A previous report established that pancreatic DNase I has only one sugar chain at Asn18 [Liao, Salnikow, Moore and Stein (1973) J. Biol. Chem. 248, 1489–1495]. We found, however, that bovine DNase I expressed in COS-1 cells was glycosylated about 70% at Asn106 in addition to being completely glycosylated at Asn18. Glycosylation of Asn106 increased to 97% when Asp107 was mutated to Glu or when Ser108 was mutated to Thr. Mutation of Asp107 to Trp had no effect, whereas a substitution with Pro at this position abolished glycosylation of Asn106. Analysis of the state of glycosylation of DNase I purified from a variety of bovine tissues revealed that DNase I from spleen, submaxillary gland, lung and adrenal had two sugar chains, whereas enzyme from pancreas and kidney had only one sugar chain. These findings demonstrate a major difference in the ability of various tissues to utilize N-linked glycosylation signals that contain suboptimal residues in the second and third positions.

scholarly journals Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4097-4104 ◽  
Author(s):  
T Misaizu ◽  
S Matsuki ◽  
TW Strickland ◽  
M Takeuchi ◽  
A Kobata ◽  
...  
Keyword(s):  
Whole Body ◽  
Renal Handling ◽  
Sugar Chain ◽  
Erythroid Progenitor ◽  
Human Erythropoietin ◽  
Sugar Chains ◽  
Effective Transfer ◽  
Branched Structure

To elucidate the role of the branched structure of sugar chains of human erythropoietin (EPO) in the expression of in vivo activity, the pharmacokinetic profile of a less active recombinant human EPO sample (EPO-bi) enriched with biantennary sugar chains was compared with that of a highly active control EPO sample enriched with tetraantennary sugar chains. After an intravenous injection in rats, 125I-EPO-bi disappeared from the plasma with 3.2 times greater total body clearance (Cltot) than control 125I-EPO. Whole-body autoradiography after 20 minutes of administration indicated that the overall distribution of radioactivity is similar, but 125I-EPO-bi showed a higher level of radioactivity in the kidneys than control 125I-EPO. Quantitative determination of radioactivity in the tissues also indicated that radioactivity of 125I-EPO-bi in the kidneys was two times higher than that of control 125I-EPO. The difference in plasma disappearance between 125I-EPO-bi and control 125I-EPO was not observed in bilaterally nephrectomized rats. The distribution of 125I-EPO-bi to bone marrow and spleen was similarly inhibited by simultaneous injection of excess amounts of either the nonlabeled EPO-bi or control EPO. These results indicate that the low in vivo biologic activity of EPO-bi results from rapid clearance from the systemic circulation by renal handling. Thus, the well-branched structure of the N-linked sugar chain of EPO is suggested to play an important role in maintaining its higher plasma level, which guarantees an effective transfer to target organs and stimulation of erythroid progenitor cells.

Sugar chain-binding specificity and native folding state of lectins preserved in hydrated ionic liquids

2015 ◽  
Vol 51 (54) ◽  
pp. 10883-10886 ◽  
Author(s):  
Kyoko Fujita ◽  
Miki Sanada ◽  
Hiroyuki Ohno
Keyword(s):  
Ionic Liquids ◽  
Thermal Treatment ◽  
Dihydrogen Phosphate ◽  
Sugar Chain ◽  
Long Term Storage ◽  
Sugar Chains ◽  
Term Storage ◽  
Folding State ◽  
Specific Sugar

Lectins, dissolved and stored in hydrated cholinium dihydrogen phosphate, maintained recognition and binding affinity to specific sugar chains even after thermal treatment or long-term storage.

scholarly journals Molecular Dynamics and Metadynamics Simulations of the Cellulase Cel48F

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Osmair Vital de Oliveira
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Md Simulations ◽  
Cellulose Fibers ◽  
Mean Square ◽  
Mean Square Deviation ◽  
Wild Type ◽  
Sugar Chain ◽  
Alcohol Production ◽  
Sugar Chains

Molecular dynamics (MD) and metadynamics techniques were used to study the cellulase Cel48F-sugar. Cellulase is enzyme that breaks cellulose fibers into small sugar units and is potentially useful in second generation alcohol production. In MD simulations, the overall structure of equilibrated Cel48F did not significantly change along the trajectory, retaining root mean square deviation below 0.15 nm. A set of 15 residues interacting with the sugar chains via hydrogen bonding throughout the simulation was observed. The free energy of dissociation (ΔGdiss.) of the chains in the catalytic tunnel of Cel48F was determined by metadynamics. The ΔGdiss. values of the chains entering and leaving the wild-type Cel48F cavity were 13.9 and 62.1 kcal/mol, respectively. We also mutated the E542 and Q543 to alanine residue and obtained ΔGdiss. of 41.8 and 45.9 kcal/mol, respectively. These mutations were found to facilitate smooth dissociation of the sugar chain across the Cel48F tunnel. At the entry of the Cel48F tunnel, three residues were mutated to alanine: T110, T213, and L274. Contrary to the T110A-Cel48F, the mutants T213-Cel48F and L274-Cel48F prevented the sugar chain from passing across the leaving site. The present results can be a guideline in mutagenesis studies to improve processing by Cel48F.

scholarly journals Sequence of bovine carbonic anhydrase VI: potential recognition sites for N-acetylgalactosaminyltransferase

1996 ◽  
Vol 318 (1) ◽  
pp. 291-296 ◽  
Author(s):  
Weiping JIANG ◽  
Joseph T WOITACH ◽  
Dwijendra GUPTA
Keyword(s):  
Amino Acids ◽  
Submaxillary Gland ◽  
Cdna Clones ◽  
Carbonic Anhydrases ◽  
Active Site Residues ◽  
Glycoprotein Hormone ◽  
Bovine Carbonic Anhydrase ◽  
Glycosylation Sites ◽  
Carbonic Anhydrase Vi ◽  
Related Proteins

Carbonic anhydrases (CAs I–VII) are products of a gene family that encodes seven isoenzymes and several CA-related proteins. We report the cloning and sequencing of the cDNA clones encoding one of these isoenzymes, CA VI, from bovine submaxillary gland. The translated polypeptide consists of 319 amino acids, including a signal peptide (14 amino acids) typical of secreted proteins. The predicted mature protein contains 305 amino acids including a 13-amino-acid C-terminal sequence that is also present in the sheep but absent in human CA VI. The deduced mature bovine protein is 87% and 68% identical to that of sheep and human CA VI, respectively. Active-site residues of the enzyme, as well as the three zinc-binding histidines and the two cysteines involved in an intra-chain disulphide bond, are all conserved in the three species. Two potential Asn-glycosylation sites are also conserved, both of which appear to be glycosylated in sheep and bovine CA VI. Two potential peptide recognition sequences are present in bovine CA VI for the glycoprotein hormone: N-acetylgalactosaminyltransferase (GalNAc-transferase), which is one of the two transferases required to form GalNAc-4-SO4 in bovine CA VI-linked oligosaccharides. Specifically, these two sequences are Asp-Leu-Lys-Met-Lys-Lys and Ile-Thr-Lys-Arg-Lys-Lys. Comparison of these sequences with sheep and human CA VI sequences indicates that distinct glycoforms of CA VI could exist in submaxillary gland from different species.

scholarly journals Location-Specific, Unequal Contribution of the N Glycans in Simian Immunodeficiency Virus gp120 to Viral Infectivity and Removal of Multiple Glycans without Disturbing Infectivity

1998 ◽  
Vol 72 (10) ◽  
pp. 8365-8370 ◽  
Author(s):  
Shinji Ohgimoto ◽  
Tatsuo Shioda ◽  
Kazuyasu Mori ◽  
Emi E. Nakayama ◽  
Huiling Hu ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Envelope Protein ◽  
Viral Infectivity ◽  
Sugar Chain ◽  
Glycosylation Sites ◽  
Immunodeficiency Virus ◽  
Cd4 Binding Site ◽  
Gp120 Subunit ◽  
Virus Recovery

ABSTRACT One of the striking features of human immunodeficiency virus, simian immunodeficiency virus (SIV), and other lentiviruses is extensive N glycosylation of the envelope protein. To assess the requirement of each N glycan for viral infectivity, we individually silenced all 23 N glycosylation sites in the gp120 subunit of SIVmac239 envelope protein by mutagenizing the canonical Asn-Xaa-Thr/Ser N glycosylation motif in an infectious molecular clone, attempted to rescue viruses from the clones, and compared the replication capability of the rescued viruses in MT4 cells. The mutation resulted in either the recovery of a fully infectious virus (category I); recovery of a faster-replicating virus, compared with the parental virus (category II); or no virus recovery (category III). These categorically different sites were not distributed randomly but were clustered. The sites of category I were localized largely in the N-terminal half, whereas the sites of categories II and III were localized in the C-terminal region, including the CD4 binding site, and the central part, including the C loop, respectively. To learn how far SIV can tolerate the removal of glycans, multiplex mutagenesis was also attempted. When they were appreciably distant from one another in the primary sequence, up to five sites could be silenced in combination without disturbing infectivity. On the other hand, it was difficult to silence contiguous sites. Thus, it appeared that a certain degree of sugar chain density over the local region had to be preserved. We discuss the potential utility of these variously deglycosylated mutants for clarifying the role of N glycans in SIV replication in vivo, as well as in the host response, and for designing vaccines and the generation of glycoprotein crystals.

scholarly journals Carbohydrate structure of human pancreatic elastase 1

1991 ◽  
Vol 278 (2) ◽  
pp. 505-514 ◽  
Author(s):  
P Wendorf ◽  
D Linder ◽  
A Sziegoleit ◽  
R Geyer
Keyword(s):  
Gel Filtration ◽  
Methylation Analysis ◽  
Complex Type ◽  
Pancreatic Elastase ◽  
Blood Group A ◽  
Elastase 1 ◽  
Glycosylation Sites ◽  
Group A ◽  
Sugar Chains ◽  
Pancreatic Elastase 1

Human pancreatic elastase 1 (E1) is a glycoprotein containing two potential N-glycosylation sites, one of which carries a carbohydrate moiety [Wendorf, Geyer, Sziegoleit & Linder (1989) FEBS Lett. 249, 275-278]. In order to study its glycosylation, glycoprotein isolated from post-mortem pancreas tissue of 75 donors was digested with trypsin. Oligosaccharides were liberated from resulting glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glycosaminyl)-asparagine amidase F, radiolabelled by reduction with KB3H4 and separated by h.p.l.c. and gel filtration. Major oligosaccharide alditol fractions, representing 67.8 mol% of total glycans, were characterized by methylation analysis and sequential degradation with exoglycosidases. The results revealed that about two-fifths of the partially truncated, mainly biantennary, complex-type glycans found comprised blood group A, B, Lea (or X), difucosyl A or difucosyl B determinants, which could be assigned to lactosamine antennae linked to Man(alpha 1-3)- residues of the sugar chains.

Detection of Sialic Acid Residues and Studies of Their Organization in Normal and Tumor α1-Acid Glycoproteins as Probed by Surface-Enhanced Raman Spectroscopy

1993 ◽  
Vol 47 (5) ◽  
pp. 535-538 ◽  
Author(s):  
Konstantin V. Sokolov ◽  
Nargiz E. Byramova ◽  
Larisa V. Mochalova ◽  
Alexander B. Tuzikov ◽  
Svetlana D. Shiyan ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Surface Enhanced Raman Spectroscopy ◽  
Tumor Patients ◽  
Sugar Chain ◽  
Healthy Donors ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Chain Organization ◽  
Sugar Chains ◽  
Sialylated Glycoproteins

Surface-enhanced Raman scattering (SERS) spectra of sialic acid (SA) benzyl, and methyl glycosides as well as natural sialylated glycoproteins from human cells of healthy donors and tumor patients have been analyzed in view of the fact that peripheral fragments of many bioactive glycoconjugates are SA residues. SA residues can be detected by SERS at concentrations as low as 10−6 M. The pattern of interaction of SA with the surface of a silver hydrosol involves the C-8 and C-9 hydroxy groups of SA. SERS spectroscopy is sensitive to changes in the content and type of branching of sialylated sugar chains in sialylglycoproteins. The differences in sialylated sugar-chain organization for α1-acid glycoproteins from healthy donors and tumor patients have been detected by means of SERS spectroscopy. The first example of the detection of SA residues for a suspension of living cells has been presented.

scholarly journals Dystroglycanopathy: From Elucidation of Molecular and Pathological Mechanisms to Development of Treatment Methods

2021 ◽  
Vol 22 (23) ◽  
pp. 13162
Author(s):  
Motoi Kanagawa
Keyword(s):  
Clinical Symptoms ◽  
Treatment Strategies ◽  
Chain Structure ◽  
Gene Products ◽  
Therapeutic Approaches ◽  
Causative Gene ◽  
Sugar Chain ◽  
The Central Nervous System ◽  
Sugar Chains ◽  
Abnormal Glycosylation

Dystroglycanopathy is a collective term referring to muscular dystrophies with abnormal glycosylation of dystroglycan. At least 18 causative genes of dystroglycanopathy have been identified, and its clinical symptoms are diverse, ranging from severe congenital to adult-onset limb-girdle types. Moreover, some cases are associated with symptoms involving the central nervous system. In the 2010s, the structure of sugar chains involved in the onset of dystroglycanopathy and the functions of its causative gene products began to be identified as if they were filling the missing pieces of a jigsaw puzzle. In parallel with these discoveries, various dystroglycanopathy model mice had been created, which led to the elucidation of its pathological mechanisms. Then, treatment strategies based on the molecular basis of glycosylation began to be proposed after the latter half of the 2010s. This review briefly explains the sugar chain structure of dystroglycan and the functions of the causative gene products of dystroglycanopathy, followed by introducing the pathological mechanisms involved as revealed from analyses of dystroglycanopathy model mice. Finally, potential therapeutic approaches based on the pathological mechanisms involved are discussed.

scholarly journals Isolation and partial characterization of the major sialoglycoprotein of human T-lymphoblastoid cells of a MOLT-4B cell line

1978 ◽  
Vol 175 (3) ◽  
pp. 823-831 ◽  
Author(s):  
M Saito ◽  
S Toyoshima ◽  
T Osawa
Keyword(s):  
Sialic Acid ◽  
Cell Line ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Exchange Chromatography ◽  
Total Carbohydrate ◽  
Lymphoblastoid Cells ◽  
Sugar Chain ◽  
Sugar Chains

A sialoglycoprotein with an approx. mol.wt. of 95000 was isolated from human lymphoblastoid cells of a MOLT-4B cell line, which was of human T-lymphocyte origin, by ion-exchange chromatography, affinity chromatography on a column of wheat-germ agglutinin-Sepharose and preparative slab-gel electrophoresis. The localization of this glycoprotein on the cell surface was indicated by surface labelling by the periodate/NaB3H4 and lactoperoxidase-catalysed iodination methods. Carbohydrate analyses of this glycoprotein revealed that its total carbohydrate content is 28% (w/w), and it contains fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in molar proportions 1.0:4.0:3.7:3.5:1.2:2.5, suggesting that it has two types of sugar chain, i.e. sugar chains like those of serum glycoproteins and sugar chains of the type found in mucins. Actually, alkaline borohydride treatment of this glycoprotein yielded tri- and tetra-saccharide, the latter containing 1 molecule of fucose in addition to each molecule of galactose, N-acetylgalactosamine and sialic acid. This glycoprotein bound to Ricinus communis agglutinin and concanavalin A as well as to wheat-germ agglutinin.

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