Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry.
Structural characterization of the HIV-1 envelope protein gp120 is very important for providing an understanding of the protein's immunogenicity and its binding to cell receptors. So far, the crystallographic structure of gp120 with an intact V3 loop (in the absence of a CD4 coreceptor or antibody) has not been determined. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for coreceptor binding and entry of the virus into T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated the glycosylation occupancy of gp120-OD8; 11 sites from 15 glycosylation motifs were determined as having high-mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information about protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4-gp120-OD8 crystal structure revealed a flexible V3 loop structure in which the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure.
Wang
Liwen
L
Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Qin
Yali
Y
Ilchenko
Serguei
S
Bohon
Jen
J
Shi
Wuxian
W
Cho
Michael W
MW
Takamoto
Keiji
K
Chance
Mark R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004330:Drosophila; D006031:Glycosylation; D015483:HIV Antibodies / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry
D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D007074:Immunoglobulin G; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; C514793:gp120 protein, Human immunodeficiency virus 1
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
J Biotechnol. 2003 Apr 10;102(1):61-71
12668315
J Proteome Res. 2006 Jun;5(6):1493-503
16740002
Mol Cell Proteomics. 2008 Dec;7(12):2386-98
18653769
Nature. 2005 Feb 24;433(7028):834-41
15729334
Methods Mol Biol. 2009;534:5-21
19277556
Nat Immunol. 2004 Mar;5(3):233-6
14985706
BMC Bioinformatics. 2007 Apr 20;8:133
17448237
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3942-7
12655044
J Biol Chem. 1999 Feb 12;274(7):4115-23
9933605
Annu Rev Biophys Biomol Struct. 2006;35:251-76
16689636
Nat Biotechnol. 2003 Jun;21(6):660-6
12754519
Glycobiology. 1991 Jun;1(3):257-63
1794038
Trends Biochem Sci. 2005 Oct;30(10):583-92
16126388
Mol Cell Proteomics. 2005 Aug;4(8):1189-93
15923566
Electrophoresis. 1999 Dec;20(18):3551-67
10612281
Biopolymers. 1996 Mar;38(3):305-20
8906967
Eur J Biochem. 2001 May;268(9):2620-8
11322882
Glycoconj J. 1999 Feb;16(2):109-23
10612411
Biochemistry. 2003 Nov 4;42(43):12447-54
14580189
Rev Sci Instrum. 2008 Feb;79(2 Pt 1):025101
18315323
Anal Chem. 2001 Sep 15;73(18):4530-6
11575803
J Synchrotron Radiat. 2007 May;14(Pt 3):233-43
17435298
J Virol. 2005 Jan;79(2):1154-63
15613343
Nat Struct Biol. 1995 Oct;2(10):842-6
7552705
Proteomics. 2006 Jul;6(14):3993-4015
16786490
Mol Cell Proteomics. 2003 Oct;2(10):1120-32
12966145
J Proteome Res. 2008 Jan;7(1):138-44
18072732
Science. 1998 Jun 19;280(5371):1949-53
9632396
Annu Rev Biochem. 1981;50:783-814
6791580
Nature. 1998 Jun 18;393(6686):648-59
9641677
Anal Chem. 2003 Jul 1;75(13):3256-62
12964777
Nature. 2007 Feb 15;445(7129):732-7
17301785
AIDS Res Hum Retroviruses. 2000 May 20;16(8):741-9
10826481
Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
12824332
Mol Cell Proteomics. 2005 Dec;4(12):1959-67
16332734
Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1552-7
17251352
Biochemistry. 2005 Mar 8;44(9):3166-75
15736927
J Virol. 2001 Jun;75(12):5541-9
11356961
Chem Rev. 2007 Aug;107(8):3514-43
17683160
Anal Chem. 2005 Jul 15;77(14):4549-55
16013872
Anal Bioanal Chem. 2004 Feb;378(4):1129-35
14658030
Anal Biochem. 1989 Aug 1;180(2):195-204
2510544
J Biomol Struct Dyn. 2001 Dec;19(3):405-18
11790140
Bioinformatics. 2006 Jan 15;22(2):195-201
16301204
J Mass Spectrom. 2007 Mar;42(3):370-9
17212372
J Biol Chem. 1987 Jan 15;262(2):785-94
3805008
Nature. 2003 Mar 20;422(6929):307-12
12646921
J Mol Biol. 2004 Nov 5;343(5):1195-206
15491606
Structure. 2008 Jan;16(1):38-51
18184582
Proc Natl Acad Sci U S A. 1989 Sep;86(17):6768-72
2771954
J Proteome Res. 2005 Mar-Apr;4(2):628-32
15822944
Arch Biochem Biophys. 1994 Jan;308(1):148-57
8311447
Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5283-8
11959979
Proteomics. 2008 Sep;8(18):3833-47
18780401
Nature. 1990 Jan 18;343(6255):219
2300166
J Mass Spectrom. 1999 Apr;34(4):311-29
10226360
Science. 2005 Nov 11;310(5750):1025-8
16284180
Structure. 2000 Dec 15;8(12):1329-39
11188697
Eur J Biochem. 1984 Sep 17;143(3):531-9
6434306
J Virol. 1993 May;67(5):2566-74
8474162
AIDS. 1991;5 Suppl 2:S21-33
1845049
Anal Chem. 2009 Oct 1;81(19):8141-9
19788317
Int J Radiat Biol. 2002 Feb;78(2):101-14
11779360
J Exp Med. 1991 Aug 1;174(2):407-15
1713252
AIDS Res Hum Retroviruses. 1991 Jan;7(1):3-16
2015114
Science. 1986 Dec 12;234(4782):1392-5
2431482
Trends Biochem Sci. 1994 Feb;19(2):65-70
8160267