Differential proteasomal processing of hydrophobic and hydrophilic protein regions: contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef - PubMed (original) (raw)

Differential proteasomal processing of hydrophobic and hydrophilic protein regions: contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef

Maria Lucchiari-Hartz et al. Proc Natl Acad Sci U S A. 2003.

Abstract

HIV proteins contain a multitude of naturally processed cytotoxic T lymphocyte (CTL) epitopes that concentrate in clusters. The molecular basis of epitope clustering is of interest for understanding HIV immunogenicity and for vaccine design. We show that the CTL epitope clusters of HIV proteins predominantly coincide with hydrophobic regions, whereas the noncluster regions are predominantly hydrophilic. Analysis of the proteasomal degradation products of full-length HIV-Nef revealed a differential sensitivity of cluster and noncluster regions to proteasomal processing. Compared with the epitope-scarce noncluster regions, cluster regions are digested by proteasomes more intensively and with greater preference for hydrophobic P1 residues, resulting in substantially greater numbers of fragments with the sizes and COOH termini typical of epitopes and their precursors. Indeed, many of these fragments correspond to endogenously processed Nef epitopes and/or their potential precursors. The results suggest that differential proteasomal processing contributes importantly to the clustering of CTL epitopes in hydrophobic regions.

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Figures

Fig. 1.

Fig. 1.

Map of the HIV-1-Nef protein showing CTL epitope peptides and proteasomal cleavage products. CTL epitope peptides are shown above the sequence. Black lines are those recognized by CTLs of HIV-infected subjects as recorded in the HIV Molecular Immunology Database. Red lines are the naturally processed peptides acid-eluted from Nef-transfected cells (see also Table 1). The 20S proteasomal cleavage products of recombinant HIV-1-Nef (LAI) protein are indicated as lines below the sequence. Fragments corresponding to acid-eluted HLA class I ligands are highlighted in red; fragments corresponding to their potential precursor peptides are highlighted in green. *, Epitopes identified by immunization of HLA-A2 transgenic mice with HIV-1-Nef (HXB3), with the sequences ALTSSNTA and MTYKAALDL instead of AITSSNTA and MTYKAAVDL (LAI); **, a 20-aa sequence (Nef161–180) where a CTL epitope of unknown HLA restriction was located; ***, subdominant epitope with the sequence DPEKEVLQWK (LAI sequence, DPEREVLEWR).

Fig. 2.

Fig. 2.

Length distribution of 20S proteasomal cleavage fragments from HIV-1-Nef protein. Recombinant HIV-1-Nef (LAI) protein was digested with purified 20S proteasomes, and the cleavage fragments were determined by MS (see Fig. 1). The preferred size range of MHC class I peptide ligands is highlighted in pink.

Fig. 3.

Fig. 3.

Frequencies of amino acids at the COOH and NH2 termini of the 143 Nef proteasomal cleavage fragments shown in Fig. 1. The COOH-terminal amino acids are shown as gray bars, and the NH2-terminal amino acids are shown as white bars. For the COOH-terminal amino acids, the proportion of peptides from the CTL epitope clusters is indicated by the hatched parts of the bars. The frequencies (%) of individual amino acids in the Nef protein are given below the x axis.

Fig. 4.

Fig. 4.

Hydrophobicity/hydrophilicity plots and conservation of the primary sequence of HIV-1-Nef (A) and HIV-1-Gag p17 (B). Hydrophobicity/hydrophilicity plots were generated with the algorithm HPLC/retention pH 7.4; similar plots were obtained with the algorithm of Abraham and Leo (ExPASy Molecular Biology Server,

www.expasy.org/cgi-bin/protscale.pl

). Plots created with window sizes between 15 and 21 looked almost identical. CTL epitope clusters and single CTL epitopes are represented by boxes at the top. For Nef-CTL epitopes, see Fig. 1; for Gag CTL epitopes, see the HIV Molecular Immunology Database. The variability plots (below the hydrophobicity plots) show the number of different amino acids (dots) at each position of the concensus sequences of subtypes A–D. Sequence alignment was performed by using

align

(

www2.igh.cnrs.fr/bin/alignguess.cgi

). *, a 20-aa sequence (Nef161–180) where an HLA class I-restricted epitope (of unknown type restriction) was mapped.

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