Identification of nitrated immunoglobulin variable regions in the HIV-infected human brain: implications in HIV infection and immune response

Abstract

HIV can infiltrate the brain and lead to HIV-associated neurocognitive disorders (HAND). The pathophysiology of HAND is poorly understood, and there are no diagnostic biomarkers for it. Previously, an increase in inducible nitric oxide synthase levels and protein tyrosine nitration in the brain were found to correlate with the severity of HAND.1,2 In this study, we analyzed human brains from individuals who had HIV infection without encephalitis and with encephalitis/HAND and compared them to the brains of healthy individuals. We identified the nitrated proteins and determined the sites of modification using affinity enrichment followed by high-resolution and high-mass-accuracy nanoLC-MS/MS. We found that nitrated proteins were predominantly present in the HIV-infected individuals with encephalitis, and, interestingly, the modifications were predominantly located on immunoglobulin variable regions. Our molecular model indicated potential interactions with HIV envelope proteins and changes on the heavy and light chain interface upon the nitration and nitrohydroxylation of these residues. Therefore, our findings suggest a role for these modifications in the immune response, which may have implications in disease pathogenesis.

Scheme 1. Experimental Design

Brain samples from the parietal cortex of HIV-negative controls, HIV-infected individuals without encephalitis, and HIV-infected individuals with encephalitis were immunoprecipitated with anti-3-nitrotyrosine antibody conjugated to protein G–agarose. The elution fraction with enriched nitrated proteins was trypsinized. The peptides were separated on a reverse-phase nano-LC system and analyzed by high-resolution tandem mass spectrometry.

Figure 1

Chemical structures of 3-nitrotyrosine and nitrohydroxy tryptophan. The exact location of nitro and hydroxyl groups on the tryptophan cannot be determined by mass spectrometric approaches. The anti-3-nitrotyrosine antibody has previously been shown to be capable of binding to both of these modified amino acids.

Figure 2

Number of proteins identified in the sample sets and the distribution of proteins identified exclusively in the HIV-E brain. (a) Venn diagram representation of differentiable proteins in the parietal cortex brain tissue immunoprecipitates. The majority of the proteins are found exclusively in the HIV-E sample set. (b) Distribution of proteins found in the immunoprecipitates from HIV-E samples. Ninety proteins are immunoglobulin variable regions, of which five proteins are immunoglobulins against HIV envelope proteins.

Figure 3

Venn diagram representation of nitrated and nitrohydroxylated peptides in the sample sets. The majority of the nitrotyrosine and nitrohydroxytryptophan containing peptides were identified in the tryptic digests of the immunoprecipitated proteins from the HIV-infected with encephalitis sample set.

Figure 4

Amino acid sequence alignment of immunoglobulin variable regions identified only in the HIV-E immunoprecipitates. The National Center for Biotechnology constraint-based multiple alignment tool (COBALT) was used. Highly conserved residues are in red, and mostly conserved residues are in blue. The nitrated residues, shown with an arrow, are conserved among different immunoglobulin variable regions. (a) Immunoglobulin light chain variable region sequences. (b) Immunoglobulin heavy chain variable region sequences.

Figure 5

Model of the complex between immunoglobulin light and heavy chains and the HIV envelope protein gp41 innercore obtained using the crystal structure of the neutralizing antibody as templates. (a) The cartoon of the heavy and light chains of the immunoglobulin are shown in yellow and green, respectively. The solvent-accessible surface of gp41 is drawn semitransparent and is colored according to the electrostatic potential (red, negative; blue, positive) around the cartoon of the receptor (colored in red). Nitrated Y32, Y36, Y49 and nitrohydroxylated W36 and W105 of the immunoglobulin as well as H199 and R214 of the receptor are drawn in a ball-and-stick representation. The positions of the nitro- and hydroxyl-group substitutions were chosen to minimize steric conflicts with the immunoglobulin. (b) Nitrated heavy chain Y32 may interact with H199 of gp41. (c) Heavy chain Y59 is also solvent-exposed, and its nitration may have a positive effect on gp41 binding through the favorable interaction between the nitro group and R214. (d) Light chain Y36 resides at the interface between the heavy and the light chains. Interestingly, the nitro group can be accommodated well at this position through H-bonds with heavy chain W105 and a backbone amine. (e) Light chain Y49 resides below the disordered loop (dashed line) between the heavy chain β4 and β5 strands, and nitration of this residue may affect this loop, which contains CDR3 and is critical for antigen binding.