Glycosylation of GD4 and Thy-1

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with m utant molecules with only single glycosylation sites and with a truncated form containing only the two NH 2 -terminal domains, indicate that independent processing occurs at each glycosylation site and that dom ain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

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THE INFLUENCE OF N-LINKED GLYCOSYLATION AND BINDING PROTEIN (BiP) ASSOCIATION IN THE SECRETION EFFICIENCY OF COMPLEX GLYCOPROTEINS

10.1055/s-0038-1644016 ◽

1987 ◽

Author(s):

Randal J Kaufman ◽

David G Bole ◽

Andrew J Dorner

Keyword(s):

Cho Cells ◽

Binding Protein ◽

Significant Proportion ◽

Chinese Hamster ◽

Site Directed Mutagenesis ◽

Ovary Cell ◽

Stable Complex ◽

Glycosylation Sites ◽

Secretion Efficiency ◽

Degree Of Association

We have studied the role of Binding Protein (BiP) or glucose regulated protein, GRP 78) in the processing and secretion of factor VIII (fVIII), von Willebrand factor (vWF), and tissue plasminogen activator(tPA) expressed in Chinese hamster ovary cell lines.fVIII is a 300 kDa protein which has a heavily glycosylated internal domain containing 20 clustered potential N-linked glycosylation sites.A significant proportion of the expressed fVIII is bound to BiP in the endoplasmic reticulum (ER) in a stable complex andnever secreted. Deletion of the heavily glycosylatedregion results in a lesser degree of association with BiP and increased secretion. Tunicamycin treatmentof cells producing the deleted form of fVIII resultsin stable association of the unglycosylated fVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites which are scattered throughout the molecule. vWF is transiently associated with BiP in the ER, demonstrating that CHO cells are competent to saecrete a complex glycoprotein. tPA, which contains 3 utilized N-linked glycosylation sites, exhibits low level association with BiP and is efficiently secreted. Disruptionof normal N-linked glycosylation of tPA, by site directed mutagenesis of the 3 Asn residues to Gin residues or by tunicamycin treatment of the tPA expressing CHO cells, results in reduced levels of secretion and increased association with BiP. This effect is enhanced by high levels of expression. The findings suggest that occupancy of glycosylation sites may effect protein folding and alter secretion efficiency by influencing the extent and stability of association with BiP.

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Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

The Journal of Cell Biology ◽

10.1083/jcb.132.6.1199 ◽

1996 ◽

Vol 132 (6) ◽

pp. 1199-1208 ◽

Cited By ~ 120

Author(s):

A Bartolazzi ◽

A Nocks ◽

A Aruffo ◽

F Spring ◽

I Stamenkovic

Keyword(s):

Cell Adhesion ◽

Regulatory Mechanism ◽

Cell Attachment ◽

Glycosylation Site ◽

Side Chain ◽

Site Specific ◽

Coated Substrate ◽

Glycosylation Sites ◽

Coated Surfaces

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

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Phosphorylation of the Oncofetal Variant of the Human Bile Salt-dependent Lipase

Journal of Biological Chemistry ◽

10.1074/jbc.m008658200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12356-12361 ◽

Cited By ~ 9

Author(s):

Alain Verine ◽

Josette Le Petit-Thevenin ◽

Laurence Panicot-Dubois ◽

Annick Valette ◽

Dominique Lombardo

Keyword(s):

High Pressure ◽

Bile Salt ◽

Cho Cells ◽

Phosphorylation Site ◽

Chinese Hamster ◽

Human Bile ◽

Site Specific ◽

Phosphorylation Motif ◽

Extracellular Compartment ◽

First Time

In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with32P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.

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Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme

Biochemical Journal ◽

10.1042/bj3040917 ◽

1994 ◽

Vol 304 (3) ◽

pp. 917-922 ◽

Cited By ~ 10

Author(s):

R D Basco ◽

L M Hernández ◽

M D Muñox ◽

I Olivero ◽

E Andaluz ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Thermal Inactivation ◽

Glycosylation Site ◽

Translation Product ◽

Site Specific ◽

Potential Glycosylation Site ◽

Glycosylation Sites ◽

Conformational Constraints ◽

Specific Elongation ◽

Protein Portion

Three exoglucanases (Exgs), ExgIa, ExgIb and Exg325, are secreted by Saccharomyces cerevisiae cells. They share a common protein portion with two potential glycosylation sites (sequons) but differ in the amount of N-linked carbohydrate [Basco, R.D., Muñoz, M.D., Hernández, L.M., Váquez de Aldana, C. and Larriba, G. (1993) Yeast 9, 221-234]. ExgIb contains two short oligosaccharides attached to asparagines (Asn) 165 and 325 of the primary translation product [Hernández, L.M., Olivero, I., Alvarado, E. and Larriba, G. (1992) Biochemistry 31, 9823-9831]. Exg325 carries a single, short oligosaccharide bound to Asn325 whereas ExgIa has at least one large oligosaccharide, since it has not been produced by mutant mnn9. To address the question of the origin of ExgIa, both sequons were individually mutated by substituting Gln for Asn. An ExgIa-like isoenzyme was still secreted by mutant Exg165 but not by mutant Exg325. Additional studies on sequential deglycosylation of ExgIa with endo-beta-N-acetylglucosaminidase H (endo H), the susceptibility of both oligosaccharides to the endoglycosidase, and analysis of the presence of GlcNAc at both asparagine residues after total deglycosylation with endo H, indicated that ExgIa contained two oligosaccharides, a short one bound to Asn165 and a large one bound to Asn325, and, accordingly, originated from ExgIb. The elongation of the second oligosaccharide did not result in a higher stability towards thermal inactivation or unfolding, or in an increased resistance to proteases as compared with ExgIb; however, the affinity of the enzyme towards laminarin decreased by 50%. This site-specific elongation occurred in the oligosaccharide that was less susceptible to endo H, indicating that these properties are determined by different conformational constraints.

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Gradually glycosylated protein C mutants (Arg178Gln and Cys331Arg) are degraded by proteasome after mannose trimming

Thrombosis and Haemostasis ◽

10.1160/th04-07-0403 ◽

2004 ◽

Vol 92 (12) ◽

pp. 1284-1290 ◽

Cited By ~ 4

Author(s):

Masako Nakahara ◽

Fumie Nakazawa ◽

Miwako Nishio ◽

Aya Shibamiya ◽

Shinsaku Hirosawa ◽

...

Keyword(s):

Protein C ◽

Three Dimensional ◽

Chinese Hamster ◽

Glycosylation Site ◽

Expression Vectors ◽

Dimensional Structure ◽

Mutant Proteins ◽

Accelerated Degradation ◽

Naturally Occurring ◽

Glycosylation Sites

SummaryProteins that fail to attain their correct three-dimensional structure are retained in the endoplasmic reticulum (ER) and eventually degraded within the cells. We investigated the degradation of mutant proteins, using naturally occurring protein C (PC) mutants (Arg178Gln and Cys331Arg) which lead to congenital deficiencies. Chinese hamster ovary (CHO) cells were transfected with normal or mutant expression vectors. The introduction of mutation at Asn329 of an unusual sequence Asn-X-Cys for N-linked glycosylation revealed that the mutation at Cys331, which may preclude a formation of disulfide bond with Cys345, resulted in no addition of N-linked oligosaccharides at Asn329. PC mutants with 4 glycosylation sites were gradually glycosylated in the ER, and the fourth glycosylation site is less accessible for glycosylation as reported for PC in plasma.The half lives of PC178 and PC331 mutants were about 5 and 4 h, respectively. PC mutants were degraded, but the degradation was inhibited by inhibitors for proteasome. Mannose trimming of N-linked oligosaccharides after glucose removal targeted PC mutants for degradation by proteasomes. And also the inhibition of glucose trimming immediately led to mannose trimming, resulting in the accelerated degradation of PC mutants. These degradations were inhibited by mannosidase I inhibitor, kifunensine. These results indicate that the initiation of mannose trimming by mannosidase I leads to the proteasomemediated degradation of glucose-trimmed or untrimmed PC mutants.

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Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein

Molecules ◽

10.3390/molecules24213924 ◽

2019 ◽

Vol 24 (21) ◽

pp. 3924

Author(s):

Hahm ◽

Lee ◽

Ahn

Keyword(s):

Fusion Protein ◽

Glycosylation Site ◽

Tandem Mass ◽

Vascular Endothelial ◽

Glycosylation Pattern ◽

Site Specific ◽

Glycosylation Sites ◽

Human Igg1 ◽

Endothelial Growth Factor ◽

Ionization Tandem Mass Spectrometry

A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked glycosylation sites: two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.

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The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins.

The Journal of Cell Biology ◽

10.1083/jcb.105.6.2665 ◽

1987 ◽

Vol 105 (6) ◽

pp. 2665-2674 ◽

Cited By ~ 206

Author(s):

A J Dorner ◽

D G Bole ◽

R J Kaufman

Keyword(s):

Heavy Chain ◽

Cho Cells ◽

Binding Protein ◽

Significant Proportion ◽

Chinese Hamster ◽

Stable Complex ◽

Tunicamycin Treatment ◽

Glycosylation Sites ◽

Relationship Of ◽

The Relationship

The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIII), von Willebrand Factor (vWF), and tissue plasminogen activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVIII has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule. vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein. tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency.

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OGP: A Repository of Experimentally Characterized O-Glycoproteins to Facilitate Studies on O-Glycosylation

10.1101/2020.03.03.975755 ◽

2020 ◽

Author(s):

Jiang-Ming Huang ◽

Meng-Xi Wu ◽

Yang Zhang ◽

Si-Yuan Kong ◽

Ming-Qi Liu ◽

...

Keyword(s):

Experimental Methods ◽

Glycosylation Site ◽

Site Specific ◽

Site Prediction ◽

Related Information ◽

Glycosylation Sites ◽

Precise Analysis ◽

Search Site ◽

User Friendly ◽

Different Sources

AbstractNumerous studies on cancer, biopharmaceuticals, and clinical trials have necessitated comprehensive and precise analysis of protein O-glycosylation. However, the lack of updated and convenient databases deters the storage and utilization of emerging O-glycoprotein data. To resolve this issue, an O-glycoprotein repository named OGP was established in this work. It was constructed with a collection of O-glycoprotein data from different sources. OGP contains 9354 O-glycosylation sites and 11,633 site-specific O-glycans mapping to 2133 O-glycoproteins, and it is the largest O-glycoprotein repository thus far. Based on the recorded O-glycosites, an O-glycosylation site prediction tool was developed. Moreover, an OGP-backed website is already available (http://www.oglyp.org/). The website comprises four specially designed and user-friendly modules: Statistic Analysis, Database Search, Site Prediction, and Data Submit. The first version of OGP repository and the website allow users to obtain vast O-glycoprotein related information, such as protein accession numbers, glycopeptides, site-specific glycan structures, experimental methods, and potential glycosylation sites. O-glycosylation data mining can be performed efficiently on this website, which can greatly facilitates O-glycosylation studies.

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Expression of a Novel Chimeric Truncated t-PA in CHO Cells Based on in Silico Experiments

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/108159 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 9

Author(s):

Fatemeh Davami ◽

Soroush Sardari ◽

Keivan Majidzadeh-A ◽

Mehdi Hemayatkar ◽

Farzaneh Barkhrdari ◽

...

Keyword(s):

Plasminogen Activator ◽

Cho Cells ◽

Serine Proteases ◽

Specific Activity ◽

Chinese Hamster ◽

Bleeding Risk ◽

Fibrinogen Concentration ◽

Truncated Form ◽

Rapid Clearance ◽

Kringle 2

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

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Metabolic engineering of CHO cells to prepare glycoproteins

Emerging Topics in Life Sciences ◽

10.1042/etls20180056 ◽

2018 ◽

Vol 2 (3) ◽

pp. 433-442 ◽

Cited By ~ 1

Author(s):

Qiong Wang ◽

Michael J. Betenbaugh

Keyword(s):

Metabolic Engineering ◽

Cho Cells ◽

Genetic Manipulation ◽

Chinese Hamster ◽

Post Translational Modification ◽

Effector Functions ◽

Recombinant Glycoproteins ◽

Production Platform ◽

Chemical Inhibitors ◽

Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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