Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers.
The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies.
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA.
Rafael S
RS
Depetris
Jean-Philippe
JP
Julien
Reza
R
Khayat
Jeong Hyun
JH
Lee
Robert
R
Pejchal
Umesh
U
Katpally
Nicolette
N
Cocco
Milind
M
Kachare
Evan
E
Massi
Kathryn B
KB
David
Albert
A
Cupo
Andre J
AJ
Marozsan
William C
WC
Olson
Andrew B
AB
Ward
Ian A
IA
Wilson
Rogier W
RW
Sanders
John P
JP
Moore
D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; D054299:env Gene Products, Human Immunodeficiency Virus