Complex N-linked glycans on Asn-89 of Kaposi sarcoma herpes virus-encoded interleukin-6 mediate optimal function by affecting cytokine protein conformation.
Kaposi sarcoma-associated herpesvirus-encoded interleukin-6 (vIL-6) and its human cellular homologue (huIL-6) share similar biological functions. Our previous work showed that N-linked glycosylation was required for optimal function of vIL6 but not huIL-6 (1). Here we describe heterogeneity in the composition of the glycans of the two N-linked sites of vIL-6. The Asn-89 site of vIL-6, found to be required for optimal cytokine function, is composed of complex glycans. The Asn-78 site is composed of high mannose glycans, which are dispensable for cytokine function. N-Linked glycosylation at the Asn-89 site was required for intracellular production of functional vIL-6, but endoglycosidase-mediated removal of N-linked glycans from secreted vIL-6 did not impair protein function. With the use of a conformation-specific antibody and tryptic digestion assays, we showed that glycosylation at the Asn-89 site of vIL-6 affected protein conformation. Human IL-6, but not vIL-6, requires IL-6Ralpha for binding to gp130. We tested the hypothesis that the Asn-89 complex glycan of vIL-6 alone was sufficient to confer binding to gp130 independently of IL-6Ralpha. Two mutants of huIL-6, made to contain additional complex N-linked glycans in the region that interacts with IL-6Ralpha, did not confer binding to gp130 independently of IL-6Ralpha. Our findings support the conclusion that complex glycans on Asn-89 of vIL-6 specifically promote a protein conformation that allows the viral cytokine to bind gp130 independently of IL-6Ralpha.
Dela Cruz
Charles S
CS
Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, Connecticut 06520, USA.
Viswanathan
Srinivas R
SR
El-Guindy
Ayman S
AS
Shedd
Duane
D
Miller
George
G
D000914:Antibodies, Viral / Q000494:pharmacology; D045744:Cell Line, Tumor; D050822:Cytokine Receptor gp130 / Q000235:genetics / Q000378:metabolism*; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D019947:Receptors, Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000914:Antibodies, Viral; C508600:IL6 protein, human; C508603:IL6R protein, human; C494199:IL6ST protein, human; D015850:Interleukin-6; D011134:Polysaccharides; D019947:Receptors, Interleukin-6; D014764:Viral Proteins; C120140:interleukin-6, RRV-HHV-8; D050822:Cytokine Receptor gp130; D005959:Glucosidases
J Mol Biol. 2004 Jan 9;335(2):641-54
14672670
J Virol. 2002 Apr;76(7):3168-78
11884541
Ann Intern Med. 1998 Jan 15;128(2):127-37
9441573
J Virol. 2001 Sep;75(18):8660-73
11507211
Eur J Biochem. 2000 Jun;267(12):3604-12
10848977
Blood. 2002 Sep 1;100(5):1919-21
12176919
Hum Immunol. 1999 Oct;60(10):921-7
10566591
Blood. 1999 Oct 15;94(8):2871-9
10515891
J Virol. 2006 Oct;80(19):9811-21
16973585
J Virol. 2009 Jan;83(2):722-33
18987143
Science. 2003 Jun 27;300(5628):2101-4
12829785
Curr Opin Cell Biol. 2001 Aug;13(4):431-7
11454449
Adv Virus Res. 2006;68:159-91
16997012
J Biol Chem. 1997 Aug 1;272(31):19625-31
9235971
Science. 2001 Mar 16;291(5511):2150-5
11251120
FEBS Lett. 2000 Jun 30;476(1-2):38-41
10878246
Clin Cancer Res. 2000 Mar;6(3):1180-9
10741750
Protein Sci. 1997 May;6(5):929-55
9144766
Science. 1996 Dec 6;274(5293):1739-44
8939871
J Exp Med. 2004 Feb 16;199(4):503-14
14970177
Blood. 2001 Nov 15;98(10):3042-9
11698289
Nat Rev Immunol. 2003 Jan;3(1):36-50
12511874
EMBO J. 1997 Mar 3;16(5):989-97
9118960
Nat Med. 1997 Mar;3(3):287-92
9055855
Annu Rev Immunol. 1997;15:797-819
9143707
J Virol. 2009 May;83(10):5117-26
19264784
Science. 2002 Nov 15;298(5597):1432-5
12434062
Front Biosci. 2007 Jan 01;12:265-81
17127298
Blood. 1998 Mar 15;91(6):1858-63
9490667
J Biol Chem. 2004 Dec 10;279(50):51793-803
15258150
J Virol. 2002 Nov;76(22):11199-208
12388679