Molecular architecture of the cleavage-dependent mannose patch on a soluble HIV-1 envelope ...

41 Pages·2016·7.22 MB·English

JVI Accepted Manuscript Posted Online 2 November 2016
J. Virol. doi:10.1128/JVI.01894-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Molecular architecture of the cleavage-dependent mannose
patch on a soluble HIV-1 envelope glycoprotein trimer

Anna-Janina Behrens1, David J. Harvey1, Emilia Milne1, Albert Cupo2, Abhinav Kumar1, Nicole D
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Zitzmann1, Weston B. Struwe1, John P. Moore2, Max Crispin1,# n
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1Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South
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Parks Road, Oxford OX1 3QU, UK p
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2Department of Microbiology and Immunology, Weill Cornell Medical College, New York, /jv
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New York, NY 10021, USA
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1 #To whom correspondence should be addressed, Max Crispin (Email: rg
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2 [email protected], Tel: [phone]) o
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Running title: Cleavage-dependent glycosylation of HIV-1 Envelope e
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5 Abstract
6 The formation of a correctly folded and natively glycosylated HIV-1 viral spike is dependent
7 on protease cleavage of the gp160 precursor protein in the Golgi apparatus. Cleavage
8 induces a compact structure, which not only renders the spike capable of fusion but also
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9 limits further maturation of its extensive glycosylation. The redirection of the glycosylation w
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10 pathway to preserve underprocessed oligomannose-type glycans is an important feature in a
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11 immunogen design as glycans contribute to or influence the epitopes of numerous broadly d

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12 neutralizing antibodies. Here, we compare quantitative site-specific analysis of a m

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13 recombinant, trimeric mimic of the native HIV-1 viral spike (BG505 SOSIP.664) to the tp
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14 corresponding uncleaved pseudotrimer and the matched gp120 monomer. We present a jv
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15 detailed molecular map of trimer-associated glycan remodelling which forms a localised m
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16 subdomain of the native mannose patch. The formation of native trimers is a critical design g
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17 feature in shaping the glycan epitopes presented on recombinant vaccine candidates. n

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18 Importance em
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19 The envelope spike of the human immunodeficiency virus (HIV-1) is a target for antibody- 9
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20 based neutralization. In some patients infected with HIV-1, highly potent antibodies have 0
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21 been isolated that can neutralize a wide range of circulating viruses. It is a goal of HIV-1 b
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22 vaccine research to elicit these antibodies by immunization with recombinant mimics of the u
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23 viral spike. These antibodies have evolved to recognize the dense array of glycans that coat
24 the surface of the viral molecule. We show how the structure of these glycans are shaped by
25 steric constraints imposed upon them by the native folding of the viral spike. This
26 information is important in guiding the development of vaccine candidates.
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27 Introduction
28 The trimeric envelope (Env) glycoprotein spike of the human immunodeficiency virus 1 (HIV-
29 1) plays a crucial role in mediating host cell infection. Its exposed position on the virus
30 surface also makes it the target for potent, broadly neutralizing antibodies (bNAbs) that are
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31 produced in a subset of infected individuals and that now influence the design of Env w
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32 immunogens intended to induce similar antibody specificities. Env is extensively a
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33 glycosylated, with glycans contributing to a significant proportion of the glycoprotein’s mass d

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34 (1). The heavily glycosylated surface of Env has been referred to as the “silent face” with the m

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35 glycans acting to shield the underlying viral protein surface from immune surveillance (2-4). tp
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36 The glycan shield constantly evolves to protect the virus from newly produced neutralizing jv
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37 antibodies. However, compared to the highly diverse protein component of Env, many of m
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38 the potential N-glycosylation sites (PNGSs) are well conserved; their total number has also g
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39 remained relatively constant despite years of viral evolution (5, 6). While derived from the n

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40 host cell’s glycosylation machinery, the high density of glycans leads to a large abundance of c
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41 underprocessed, oligomannose-type glycans (Man5-9GlcNAc2) (7-11). The glycan shield is, be
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42 therefore, surprisingly homogenous given the extent of glycosylation, and it is itself a target 1
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43 for bNAbs that recognize exclusively glycan or mixed glycan/protein epitopes (12-25). 0
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44 The Env spike is a trimer of heterodimers composed of the two subunits gp41 and y

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45 gp120, which are generated when a protease, usually furin, cleaves the pro-protein gp160 e
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46 glycoprotein (26-31). Furin most probably cleaves gp160 in the trans Golgi network (TGN),
47 but it is able to cycle between the TGN, the endosomal compartments and the cell surface
48 and is also believed to be able to act in the early secretory pathway (27, 32, 33). Furin is an
49 important factor in Env immunogen production as it is now commonly co-expressed to
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50 generate fully cleaved, recombinant, soluble trimers that structurally mimic native Env (34,
51 35). In contrast, structural and antigenic analyses of many uncleaved Env constructs show
52 that they adopt non-native conformations, with gp120 subunits dangling from a gp41 core,
53 unless additional modifications are made to overcome the defect (36-39). Cleavage is,
54 therefore, important for the structural integrity of the trimer, which in turn greatly D
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55 influences how the trimer is glycosylated (39, 40). n
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56 Several studies on monomeric gp120 proteins have demonstrated the presence of a e
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57 cluster of oligomannose-type glycans, the ‘intrinsic mannose patch’ (IMP), which arises o
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58 because the glycan shield creates steric constraints that restrict the actions of ER and Golgi t
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59 α-mannosidases (7, 8, 11, 41-43). The IMP is localized at the outer domain of gp120 and /jv
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60 comprises the epitopes of numerous glycan-influenced bNAbs (15, 21, 23, 24, 44-47). The s
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61 IMP is a characteristic feature of gp120 that is conserved across all HIV-1 clades (7, 8, 48) o
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62 and longitudinally during infection (49). Analyses of recombinant soluble, native-like trimers o
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63 (40, 50), membrane-associated trimers (51) and virion-derived Env (7-9) all show that their e
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64 oligomannose-type glycan contents are even higher than for gp120 monomers, implying m
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65 that a ‘trimer-associated mannose patch’ (TAMP) exists (52). However, the glycan sites r
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66 forming any such TAMP have not previously been defined. 2
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67 Cleaved, soluble native-like Env trimers are currently one focus of structure-guided b
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68 immunogen design programs. A detailed site-specific N-glycosylation analysis of the u
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69 prototype of this class of trimers, BG505 SOSIP.664, has revealed fine structural details of
70 the glycan shield (35, 50, 53). The gp120 subunit of this trimer is dominated by
71 underprocessed Man8-9GlcNAc2 glycans, which form a mosaic of densely organized
72 oligomannose clusters on the outer domain and the trimer interface (40, 50). In contrast,
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73 glycosylation sites with a mixed processing state (i.e., sites containing both highly processed,
74 complex and oligomannose-type glycans) are found at the trimer apex and near its N- and C-
75 termini (50). Compact folding seems to be major driver behind the oligomannose-
76 dominated profile of the glycan shield on native trimers. For example, uncleaved, non-
77 native constructs that adopt a much more open and irregular conformation have a D
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78 significantly reduced content of oligomannose-type glycans, because the now more n
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79 accessible structures are efficiently processed (39, 40). We refer here to these uncleaved, d
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80 non-native glycoproteins as pseudotrimers, to reflect their lack of the three-fold symmetry fr
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81 that is a characteristic feature of the structures of natively folded trimers (54, 55).
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82 Here, we reveal the detailed effect of cleavage-induced, native-like trimerization on /jv
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83 N-glycan processing. Our quantitative, site-specific analysis of a set of comparably expressed s
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84 and purified monomeric (i.e., gp120), uncleaved pseudotrimeric and fully cleaved trimeric o
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85 envelope glycoproteins of the same genotype (i.e., BG505) identifies the location of the o
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86 cleavage-dependent TAMP. Thus, the glycans that form the TAMP are found in regions near e
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87 the trimer apex and the protomer interface. We show that, compared to pseudotrimers, m
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88 these regions are even more highly processed on monomeric gp120, implying that the r
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89 oligomerization events impose additional regional restrictions on glycan processing. This 2
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90 finding is echoed across the panel of glycoproteins in the analysis of O-linked glycosylation 8

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91 at T499 near the trimer base, which is present at almost unmeasurable levels on native-like g
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92 trimers. We are also able to classify the glycan processing state of all N-glycans present on s
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93 the various constructs, thus extending our earlier site-specific analysis of the BG505
94 SOSIP.664 trimer (50). Our findings emphasize how the glycan shield is influenced by the
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95 quaternary structure of the Env spike, and highlight the relevance of native-like structures
96 to Env vaccine development.
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98 Materials and Methods D
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99 Expression and purification of Env proteins w
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100 The design of the BG505 SOSIP.644 and WT.SEKS constructs has been described elsewhere d
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101 (35, 36). The BG505 gp120 monomer sequence was identical to the gp120 subunit of the fr
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102 SOSIP.664 trimer, including the T332N and A501C mutations. Each env gene was transiently
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103 expressed in human embryonic kidney (HEK) 293F cells using the pPPI4 vector system as :
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104 described elsewhere (35, 56). A furin-expression plasmid was co-transfected with the i.a
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105 SOSIP.664 construct to increase trimer cleavage efficiency (34, 56). Env proteins were .
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106 isolated from culture supernatants by 2G12-affinity chromatography, followed by size- /
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107 exclusion chromatography (SEC), as described previously (35, 57). D
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108 Analysis of total glycan profiles by HILIC-UPLC m
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109 N-linked glycans were enzymatically released from Env proteins by in-gel digestion with 9
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110 Peptide-N-Glycosidase F (PNGase F) and subsequently fluorescently labelled with 2- 0
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111 aminobenzoic acid (2-AA), as previously described (40, 58). The 2-AA labelled glycans were b
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112 subsequently analysed by hydrophilic interaction chromatography-ultra performance liquid u
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113 chromatography (HILIC-UPLC) in a Waters Acquity UPLC instrument (40). The abundance of
114 oligomannose-type glycans was assessed by digesting the released glycans with Endo H
115 (New England Biolabs) (40).
116 Site-specific N-glycosylation analysis
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117 Ion mobility MS using a Waters Synapt G2Si mass spectrometer was used to analyze the
118 total pool of PNGase F-released glycans from the Env glycoproteins, as described previously
119 (50). The results were used to create sample-specific glycan libraries for the subsequent
120 site-specific N-glycosylation analyses. Env proteins (100-150 µg) were denatured and
121 alkylated as described previously (50). The following proteases or combinations of proteases D
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122 were used for digestion at a 1:30 (w/w) ratio for 12 h at 37 °C, according to manufacturers’ n
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123 instructions: Trypsin, chymotrypsin, GluC (all Mass Spectrometry Grade, Promega) and d
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124 pronase (Sigma-Aldrich). We also performed a sequential digestion using GluC (4 h, 37 °C) fr
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125 and then trypsin (12 h, 37 °C). Protease-digested samples were enriched for glycopeptides
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126 using the ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore). :
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127 Additionally, deglycosylated Env samples were prepared by digestion of tryptic and s
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128 chymotryptic (non-glycopeptide enriched) peptides with Endo H (4 h, 37 °C), followed by o
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129 digestion with PNGase F (4 h, 37 °C), according to the manufacturer’s instructions. The o
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130 processing state of the glycan site was then classified according to their susceptibility to e
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131 Endo H and PNGase F. The basis of this assignment method is that Endo H cleaves m
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132 oligomannose-type and hybrid glycans but leaves a single GlcNAc moiety that is not r
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133 removed by the subsequent PNGase F digestion (leads to deamidation at sites with 2
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134 complex-type glycans). Unoccupied PNGSs remain as unmodified asparagines. Deamidation 8

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135 of asparagines can also occur spontaneously as a non-enzymatic reaction in proteins and g
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136 peptides (59), making this method unsuitable for exact quantifications. The information s
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137 gained by this type of analysis is, however, sufficient for glycan classification into broad
138 processing states.
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139 All samples were analysed by liquid chromatography-electrospray mass
140 spectrometry (LC-ESI MS) on a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher
141 Scientific), as described previously (50). Data interpretation and quantification was
142 performed using ByonicTM and ByologicTM software (50).
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143 Site-specific O-glycosylation analysis o
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144 We assessed O-glycosylation by using ByonicTM and ByologicTM software to analyze LC-ESI nlo
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145 MS spectra derived from tryptic-digested, deglycosylated Env proteins, searching for the e
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146 most common human O-glycans. Any exact mass matches of O-glycosylated peptides were r
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147 manually confirmed by seeking the characteristic oxonium ions, as well as peptide h
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148 fragmentation ions, among the HCD fragmentation data. The relative abundances of :/
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149 individual O-glycoforms and the corresponding unmodified peptide were determined by a
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150 summing the intensities of the extracted-ion chromatograms (XICs) over all charge states. .o
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151 Relative abundance = [Intensity of one glycoform] / [Intensities of all glycoforms identified o
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152 on the same peptide].
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154 Results
155 Glycosylation processing on monomeric, uncleaved and cleaved Env proteins
156 To thoroughly investigate the influence of trimerization- and cleavage-induced structural
157 integrity on Env glycosylation, we used comparable conditions to express and purify
158 monomeric gp120, native-like SOSIP.664 trimers and uncleaved, non-native WT.SEKS gp140 D
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159 pseudotrimers, all based on the BG505 genotype. The design and characteristics of these
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160 proteins have been described previously (35, 53, 54, 56, 60-62). The WT.SEKS construct a
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161 differs from SOSIP.664 in that it lacks the stabilizing SOSIP mutations and contains an
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162 inactivated furin cleavage site (RRRRRR mutated to SEKS); the resulting uncleaved

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163 pseudotrimers have a non-native conformation (36). The monomeric gp120 protein is p
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164 isogenic with the gp120 subunit of the SOSIP.664 trimer. The designs of all three constructs i.
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165 are summarized in Figure 1A. The three proteins were all produced in transiently
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166 transfected HEK 293F cells and purified using 2G12 affinity chromatography, followed by g
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167 size exclusion chromatography.
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168 N-linked glycans were enzymatically released from all three constructs, fluorescently m
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169 labelled and analyzed by HILIC-UPLC (Figure 1B). Using Endo H to digest the oligomannose- e
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170 type glycans and by integration of the resulting chromatograms, oligomannose-type glycans ,

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171 were shown to account for 36% (gp120 monomers), 35% (WT.SEKS gp140) and 68% 1
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172 (SOSIP.664 trimers) of the total glycan populations. This outcome is consistent with a y

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173 previous report that complex-type glycans are significantly more abundant on uncleaved e
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174 pseudotrimers than on native-like trimers (40). Although the analyses of the WT.SEKS and
175 SOSIP.664 glycoproteins include contributions from the four glycans on gp41, the overall
176 outcome is dominated by the 24 glycan sites on gp120. In summary, the total oligomannose
177 contents of both the monomeric gp120 and uncleaved gp140 forms of BG505 Env are quite
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178 similar, with each protein having approximately half the oligomannose content of the
179 native-like SOSIP.664 trimers.
180 Glycan databases reveal details of cell-directed glycan processing
181 To facilitate the subsequent site-specific N-glycosylation analysis of the gp120, WT.SEKS and
182 SOSIP.664 glycoproteins, and to probe their glycan structures in greater detail, we D
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183 generated libraries of enzymatically released, unlabeled glycans using ion-mobility- n
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184 electrospray ionization mass spectrometry (IM-ESI MS). The mobility-extracted singly d
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185 charged glycans from the three glycoproteins are shown in Figure 2. A significant population fr
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186 of oligomannose-type Man5-9GlcNAc2 glycans is present on all three glycoproteins and is h
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187 most evident in the BG505 SOSIP.664 spectrum. This observation is consistent with the :/
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188 HILIC-UPLC data presented in Figure 1. Ion mobility mass spectrometry using negative i.a
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189 fragmentation mode allows the detailed assignment of isomeric glycan structures (63). The .
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190 number and type of glycan structures identified for the gp120, WT.SEKS and SOSIP.664 /
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191 proteins are with about 90 identified isobaric structures highly similar, although the D
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192 abundances vary (Table S1). Fine structural details of the diversity of complex-type glycans m
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193 present on all three constructs were revealed. The extremes of glycan processing, including r

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194 the presence of terminal fucoses, sulfates, GlcNAcs and GalNAcs, are directed by the ,

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195 producer cell’s glycosylation machinery. In contrast, the oligomannose-type glycans 1
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196 represent an intrinsic structural feature of Env (40). y

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197 Quantitative site-specific N-glycosylation analysis s
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198 We performed a quantitative, site-specific N-glycosylation analysis using in-line LC-ESI MS,
199 to thoroughly investigate how the formation of native-like, cleavage-induced trimers
200 influences the glycan shield. We have previously validated this method (50). We used a
201 panel of different proteases to create peptides and glycopeptides from the gp120, WT.SEKS
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