Quoc V. Nguyen

Inhibition by leupeptin and antipain of the intracellular proteolysis of Ii

Intracellular cleavage of Ii was evaluated in immunoprecipitates of radiolabeled Raji cells treated with protease inhibitors (leupeptin, antipain, chymostatin, and pepstatin) or blockers of endosomal function (chloroquine and monensin). Immunoprecipitates with anti-class II and anti-Ii(12-28) sera and VIC-Y1 MoAb revealed Ii cleavage products of 21,000 and 10,000 daltons (p21 and p10) only in leupeptin- and antipain-treated cells. Both p21 and p10 were judged to be N-terminal products because they were recognized with anti-Ii(12-28) and not with anti-Ii(183-193) or anti-Ii(192-211) sera. p10 might be derived from p21 because its intensity was increased in inverse proportion to p21 as a function of leupeptin or antipain concentration. p21, but not p10, was recognized by anti-class II antibody and thus might originate from class II-associated Ii. In pulse-chase studies, p21 and p10 appeared at 2 hr and later after Ii synthesis. p25, an Ii C-terminal fragment, was about 60% reduced by leupeptin or antipain. Intracellular proteolytic cleavage of class II-associated Ii appeared to follow two pathways leading either to N-terminal p21 and p10 or to C-terminal p25. Such cleavages might regulate or catalyze foreign antigen binding to class II.

Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1.

We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection.

Effects of brefeldin A on cleavage of invariant chain to p21 and p10 and the appearance of Ii-freed class II MHC dimers

Intracellular cleavage of class II MHC-associated Ii to p21 and p10 and the appearance of Ii-freed alpha, beta dimers were concurrent events lasting from 1 to 6 hr after synthesis of alpha, beta, Ii trimers, possibly related to charging of foreign peptides to the class II MHC antigen-binding site. Sequential immunoprecipitations of pulse-chase radiolabeled cells were made four times with anti-Ii monoclonal antibody to remove Ii and alpha, beta, Ii trimers and then with anti-class II antibody to detect the time-dependent appearance of Ii-freed alpha, beta dimers. The cleavage of Ii to p21 and p10 was revealed in leupeptin-treated cells. Cell treatment with Brefeldin A (BFA) was associated with a decrease in Ii-freed alpha, beta dimers, with inhibition of leupeptin-revealed cleavage of Ii to p21 and p10, and with persistence of endoglycosidase H susceptibility of Ii and class II alpha, beta chains. We conclude that in untreated cells, cleavage and release of Ii from class II MHC alpha and beta chains occur after those complexes traverse a BFA-sensitive step in the Golgi apparatus.

Intracellular synthesis of Epstein-Barr virus membrane antigen gp350/220. Inhibitory effect of monensin on its expression.

We have defined the intracellular expression and localization of gp350/220, one of the Epstein-Barr virus (EBV) induced membrane antigens, on 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and n-butyrate-treated P3HR-1 cells. 1B6 monoclonal antibody (mAb) immunoprecipitated gp350/220 from [35S]-methionine-labeled cells, as confirmed with other mAbs (2L10, 72A1, and C1), to the same membrane antigen. The appearance of gp350/220 was observed about 14 h after TPA and n-butyrate activation and reached a maximal level at about 48 h. 1B6 mAb membrane immunofluorescence-positive and cytoplasmic fluorescence-positive cells appeared progressively in cell populations at the same frequencies. Cytoplasmic immunofluorescent staining with 1B6 mAb demonstrated a paranuclear complex which was identical to a rhodamine-labeled wheat germ agglutinin-stained pattern which has been ascribed to the Golgi apparatus. We investigated the effect of monensin on gp350/220 expression and processing. Monensin at 10(-7) M significantly inhibited membrane antigen expression in the Golgi apparatus and on the cell surface, but had a negligible effect on synthesis of viral capsid antigen, early antigen, and viral DNA. The inhibition of gp350/220 with monensin was further characterized by the immunoprecipitation of gp350/220 with anti-MA-positive human sera and mAbs. Monensin treatment resulted in the accumulation of a 165-kD molecule which was judged to be a precursor of gp350/220. These results were consistent with the view that the Golgi apparatus plays an important role as a place of synthesis, processing, and maturation of gp350/220.

Characterization of the invariant chain C-terminus (Glu183-Glu193) epitope which is obscured in processed Ii, MHC α,β trimers☆

The E1 serum was developed against invariant chain peptide Ii(183–193) in order to study the function of the Ii protein which associates with class II MHC α,β chains from time of synthesis until cleavage and release, possibly regulating the binding of antigenic peptides. Subpopulations of Ii, Ii(VIC) and Ii(E1), respectively, were demonstrated by sequential immunodepletions and immunoprecipitations with: (1) VIC-Y1 monoclonal antibody to an N-terminal epitope of Ii, and (2) E1 rabbit antiserum to Ii(183–193). In 3hr radiolabeled cells, VIC-Y1 recognized Ii, Ii and N- and O-linked glycosylation (IpN, IpO), p41 and co-precipitated class II α,β chains, while E1 recognized Ii, IpN and immature Ii-α complex. In 15 min radiolabeled cells, each antibody recognized similar, immature Ii forms without α,β. Urea denaturation of Ii(VIC) rendered the main Ii species but not IpO immunoprecipitable with E1. E1 recognized O-glycanase-treated Ii(VIC). We conclude that the Ii(183–193) epitope was obscured by interactions of Ii with class II α,β chains and by the O-linked glycosylation of Thr187, which may in part regulate association of Ii to class II α and β chains.

Roles of Accessory Molecules in Processing and Presentation of Foreign Antigens

The Ii sequence Phe146-Val164 was hypothesized to coil as an amphipathic, α helix in the desetope of class II MHC antigens until release in an acidic, foreign antigen-containing endosome to catalyze charging of the desetope with a structurally similar foreign peptide (1). A serum from one of four rabbits injected with a KLH-conjugated, synthetic peptide of Ii sequence 146–169, substituting Tyr for Phe146, immunoprecipitated a 67-kD protein from Raji cells after 3 hours [35S]methionine labeling and 69- and 67 kD-proteins after 5 or 10 hours of labeling, respectively. The 67-kD protein was not sensitive to endoglycosidase F treatment or tunicamycin and had a pI about 5.5. p67 was not surface expressed as judged by immunofluorescence analyses with the antiserum and by immunoprecipitation of surfacebiotinylated proteins. These human molecules might correspond to the murine proteins p72/74, described by Lakey et al. (2) to have a potential role in antigen presentation.

Structures of Class II MHC Molecules and Accessory Proteins During Trafficking in Subcellular Compartments

Synthetic rates of class II major histocompatibility complex (MHC) and associated molecules increased appreciably after 4 to 8 hours of polyclonal activation without any perceptible change in forms of those molecules. Ii was cleaved in intracellular vesicles to release a COOH-terminal p25 fragment at 20 to 40 minutes after [35S] methionine labeling. The association of Ii to class II MHC antigen could protect the desetope of class II MHC molecules from ambient peptides until reaching a foreign peptide containing endosome, there catalyzing the charging of digested foreign peptides onto the desetope in a concerted mechanism of Ii release and association of the foreign peptide. Ii could also retain class II MHC antigens in an endosome until charged for transport to the cell surface.

Differential Tyrosine Phosphorylation of LAT Associated with Engagement of CD3, MHC Class I and Class II

Differential Tyrosine Phosphorylation of LAT Associated with Engagement of CD3, MHC Class I and Class II

Regulation of Antigen Presentation in Human Lymphocytes by Invariant Chain CLIP and Its Adjacent N-terminal Peptides 36

We used five overlapping peptides comprising the N-terminal sequence 76-103 of the invariant chain (Ii) in mixed lymphocyte reaction (MLR) and antigen presentation system specific for tetanus toxoid (TT) to study their effects on human T cell proliferation. Invariant chain plays important role in the transport of major histocompatibility class II (MHC class II), and along with HLA-DM, the editing and charging of antigenic peptide onto the antigen binding groove of MHC class II.

Inhibition of Epstein-Barr Virus Release by anti-MA Antibody

We have previously reported a murine monoclonal antibody (MAb 1B6) which recognized an Epstein-Barr virus (EBV) membrane antigen (MA) and blocked release of infectious EBV from virus-producing P3HR-1 cells.