scholarly journals Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-γ

1994 ◽  
Vol 303 (3) ◽  
pp. 831-840 ◽  
Author(s):  
T Sareneva ◽  
J Pirhonen ◽  
K Cantell ◽  
N Kalkkinen ◽  
I Julkunen
Keyword(s):  
Interferon Γ ◽  
Glycosylation Site ◽  
Biologically Active ◽  
Human Interferon ◽  
Mutant Form ◽  
Ifn Gamma ◽  
Glycosylation Sites ◽  
Mutant Forms ◽  
Kinetics Of

Human interferon-gamma (IFN-gamma) is a secretory glycoprotein, which has two potential N-linked glycosylation sites at positions Asn-25 and Asn-97 of its 143 amino acid long mature polypeptide chain. In order to understand the role of glycan residues in the synthesis and secretion of human IFN-gamma, both or either one of the potential N-linked glycosylation sites were mutated to Gln. The mutant and the wild-type (Wt) polypeptides were expressed in insect cells using a baculovirus vector. Elimination of the N-glycosylation site at position Asn-97 (N97Q) resulted in secreted protein yields of 70-90% as compared with the Wt production, whereas only 10-25% (N25Q) and 1-10% (N25Q,N97Q) levels of protein production was observed when the first or both sites were mutated, respectively. Although there was a difference between extracellular levels of produced protein, the kinetics of secretion was similar for all different IFN-gamma molecules. The Wt and the N-glycosylation site mutants were all secreted as dimers. The formation of biologically active dimers was more efficient for IFN-gamma polypeptides that had the intact glycosylation site at Asn-25 as compared with the other two mutant forms of IFN-gamma. The extent of dimerization correlated well with the observed secretion. The specific antiviral activity was of the same order (1 x 10(7) i.u./mg of protein) for the glycosylated IFN-gamma molecules, whereas it was slightly lower (0.5 x 10(7) i.u./mg of protein) for the unglycosylated mutant form.

scholarly journals N-glycosylation of human interferon-γ: glycans at Asn-25 are critical for protease resistance

1995 ◽  
Vol 308 (1) ◽  
pp. 9-14 ◽  
Author(s):  
T Sareneva ◽  
J Pirhonen ◽  
K Cantell ◽  
I Julkunen
Keyword(s):  
Biological Activities ◽  
Interferon Γ ◽  
Glycosylation Site ◽  
Cathepsin G ◽  
Human Interferon ◽  
Globular Structure ◽  
Ifn Gamma ◽  
Protease Resistance ◽  
Glycosylation Sites ◽  
Defective Mutant

Human interferon-gamma (IFN-gamma) is a secretory, dimeric glycoprotein that forms a compact globular structure with potential N-linked glycosylation sites at Asn-25 and Asn-97 on the surface of the dimer. In natural leucocyte IFN-gamma (nIFN-gamma), 52%, 39% and 9% of the monomers are core-glycosylated in two, one or none of the potential N-glycosylation sites respectively. Chemical cross-linking of nIFN-gamma with glutaraldehyde revealed that 4, 3, 2 or 1 glycosylation sites occupied 28%, 40%, 26% and 6% of the dimers respectively. In baculovirus-produced wild-type (Wt) and N-linked glycosylation site-defective mutant (N25Q or N97Q, Asn-25 or Asn-97 substituted by Gln) IFN-gamma proteins, the extent of core glycosylation of monomers reflected the glycan composition of dimers. This suggests that dimers are formed randomly and independently of glycosylation. The glycan residues of IFN-gamma, especially at Asn-25, play an important role in protease resistance. Unglycosylated recombinant IFN-gamma proteins (from Escherichia coli and baculovirus) and N25Q IFN-gamma were sensitive to crude granulocyte protease, purified elastase, cathepsin G and plasmin degradation. Fully glycosylated nIFN-gamma and baculovirus Wt and N97Q IFN-gamma showed full or partial resistance to these proteases. These results emphasize the importance of glycan residues, especially at Asn-25, in the proteolytic stability of human IFN-gamma. Whether the differential glycosylation of n- and recombinant IFN-gamma (rIFN-gamma) is reflected in their biological activities in tissues or their clinical applicability is not known.

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

1996 ◽  
Vol 132 (6) ◽  
pp. 1199-1208 ◽  
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.

Role of Binding Site Loops in Controlling Nitric Oxide Release:  Structure and Kinetics of Mutant Forms of Nitrophorin 4†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (21) ◽  
pp. 6679-6690 ◽  
Author(s):  
Estelle M. Maes ◽  
Andrzej Weichsel ◽  
John F. Andersen ◽  
Donald Shepley ◽  
William R. Montfort
Keyword(s):  
Nitric Oxide ◽  
Binding Site ◽  
Nitric Oxide Release ◽  
Nitrophorin 4 ◽  
Mutant Forms ◽  
Kinetics Of

scholarly journals Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3877-3882 ◽  
Author(s):  
C Sgadari ◽  
AL Angiolillo ◽  
G Tosato
Keyword(s):  
Interleukin 12 ◽  
Human Interferon ◽  
Ifn Gamma ◽  
Mouse Splenocytes ◽  
Angiogenesis Inhibition ◽  
Inhibition Of Angiogenesis ◽  
Inducible Protein ◽  
Interferon Inducible Protein

Interleukin 12 (IL-12), a multifunctional cytokine produced by macrophages and B-cell lines, induces interferon-gamma (IFN-gamma) production, stimulates growth of both T and natural killer cells, promotes Th1-type helper T-cell responses, and inhibits neovascularization. Because the human interferon-inducible protein 10 (IP-10) can also inhibit neovascularization, we tested whether IP-10, induced by IL-12 through the intermediate IFN-gamma, might be a mediator of IL-12 angiogenesis inhibition. We report here that murine IL-12 profoundly inhibited basic fibroblast growth factor (bFGF)- induced Matrigel neovascularization in vivo, and that this effect of IL- 12 was neutralized by systemic administration of antibodies to either murine IFN-gamma or IP-10. Murine IL-12 induced murine IP-10 expression in mouse splenocytes, and human IFN-gamma induced human IP-10 expression in purified human endothelial cells, suggesting that IL-12 can induce IP-10 expression in certain cells. These results document the important role of IP-10 as a mediator of angiogenesis inhibition by IL-12, and raise the possibility that IP-10 may also contribute to the antitumor effect of IL-12.

scholarly journals Human interferon-γ lacking 23 COOH-terminal amino acids is biologically active

FEBS Letters ◽  
1988 ◽  
Vol 230 (1-2) ◽  
pp. 201-204 ◽  
Author(s):  
Masao Sakaguchi ◽  
Susumu Honda ◽  
Mariko Ozawa ◽  
Osamu Nishimura
Keyword(s):  
Amino Acids ◽  
Interferon Γ ◽  
Biologically Active ◽  
Human Interferon ◽  
Terminal Amino

NON-RECEPTOR-MEDIATED TISSUE LOCALIZATION OF HUMAN INTERFERON-γ: ROLE OF HEPARAN SULFATE/HEPARIN-LIKE MOLECULES

Cytokine ◽  
1996 ◽  
Vol 8 (7) ◽  
pp. 557-566 ◽  
Author(s):  
Hugues Lortat-Jacob ◽  
Christine Brisson ◽  
Sylviane Guerret ◽  
Gérard Morel
Keyword(s):  
Heparan Sulfate ◽  
Interferon Γ ◽  
Human Interferon ◽  
Tissue Localization

scholarly journals Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2

2020 ◽  
Author(s):  
Asif Shajahan ◽  
Stephanie Archer-Hartmann ◽  
Nitin T. Supekar ◽  
Anne S. Gleinich ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Viral Entry ◽  
Glycosylation Site ◽  
Converting Enzyme ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Viral Binding ◽  
Glycosylation Sites

AbstractThe emergence of the COVID-19 pandemic caused by SARS-CoV-2 has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 human cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race, and pre-existing conditions of COVID-19 patients.

Selective removal of alpha heavy-chain glycosylation sites causes immunoglobulin A degradation and reduced secretion

1988 ◽  
Vol 8 (10) ◽  
pp. 4197-4203
Author(s):  
A K Taylor ◽  
R Wall
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin A ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Myeloma Cells ◽  
Heavy Chains ◽  
Attachment Sites ◽  
Glycosylation Sites ◽  
Kinetics Of

The importance of carbohydrate in the secretion of immunoglobulin A (IgA) has previously been suggested by results of studies with tunicamycin, which prevents N-linked glycosylation of all cell glycoproteins. To directly evaluate the role of individual oligosaccharides in the secretion of IgA, we have used site-directed mutagenesis to selectively eliminate the two N-linked attachment sites reported to be glycosylated in alpha heavy chains. Transfected wild-type and mutant alpha genes were expressed in kappa light-chain-producing MPC-11 variant myeloma cells, and secretion kinetics of the IgAs were compared. Removal of either or both glycosylation sites led to intracellular alpha heavy-chain degradation and a 90 to 95% inhibition of IgA secretion. These results reveal that both N-linked oligosaccharides of the alpha heavy chain are essential for intracellular stability and normal secretion of IgA. This suggests that the key function of carbohydrate here is to maintain proper conformation of the glycoprotein. We also found that when expressed in the MPC-11 variant cells, alpha heavy chains were glycosylated at a third, normally unused site.

scholarly journals Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Glycoproteins

2005 ◽  
Vol 79 (13) ◽  
pp. 8400-8409 ◽  
Author(s):  
Anne Goffard ◽  
Nathalie Callens ◽  
Birke Bartosch ◽  
Czeslaw Wychowski ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Glycosylation Site ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Wild Type ◽  
Virus Envelope ◽  
Glycosylation Sites

ABSTRACT Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.

Cytokine immunotrapping: an assay to study the kinetics of production and consumption or degradation of human interferon-γ

1995 ◽  
Vol 182 (2) ◽  
pp. 251-261 ◽  
Author(s):  
Ali Cezmi Akdiş ◽  
Harry Towbin ◽  
Patrice Libsig ◽  
Jutta Motz ◽  
Şefik Şanal Alkan
Keyword(s):  
Interferon Γ ◽  
Human Interferon ◽  
Production And Consumption ◽  
Kinetics Of
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