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We have bred to homozygosity gene disruptions for the transporter associated with antigen processing 1 (TAP1) and beta 2-microglobulin (beta 2m), each of which plays a distinct role in providing class I MHC subunits. Surface expression of H-2Kb or Db on cells derived from TAP1/beta 2m -/- mice was undetectable by immunofluorescence or immunoprecipitation, unlike the situation observed for TAP1 -/- and beta 2m -/- single mutant mice. Yet, TAP1/beta 2m -/- cells were able to elicit a CD8+ cytotoxic T cell (CTL) response in mice of different H-2 haplotypes and could be killed by anti-H-2b specific CTL. Furthermore, TAP1/beta 2m -/- skin grafts were rejected by bm1 mutant mice. This suggests that very low levels of conformed class I heavy chains can reach the cell surface even in the complete absence of TAP1 and beta 2m gene products, and that these molecules may select a functional CD8+ T cell repertoire. Indeed, CD4-CD8+ T cells were detected in TAP1/beta 2m -/- mice, but in numbers lower than in either of the single mutant mice. Nonetheless, it was possible to elicit a CD8+ allospecific and H-2b reactive CTL response in TAP1/beta 2m -/- mice. In line with this, TAP1/beta 2m -/- mice rapidly rejected TAP1/beta 2m +/- skin grafts. Our results suggest that some MHC class I heavy chains in TAP1/beta 2m -/- cells can reach the cell surface in a form that allows recognition by allospecific CTL and positive selection of CD8+ T cells.
The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of the serotonin transporter caused the Sf9 cells to accumulate [3H]serotonin (Km 78 nM) and to bind the specific transport inhibitor [125I]RT155 (2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane) (Kd 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies of the serotonin transporter/cell (9 pmol/mg of membrane protein). Immunoreactive bands of apparent M(r) 54,000 (unglycosylated) and 60,000 (glycosylated) were observed in Western blots of membrane proteins from infected cells. The 54-kDa band was significantly smaller than the expected M(r) of 72,500 predicted from the cDNA sequence. The 54-kDa band was shown to represent the intact serotonin transporter by expressing a recombinant serotonin transporter that contained c-Myc and FLAG epitope tags engineered at the N and C termini, respectively. Both tags were present on a membrane protein that migrated slightly slower than the previously observed 54-kDa band, consistent with the extra mass added by the tags. The tags did not affect the Kd for [125I]RT155 binding. The effect of N-linked glycosylation on ligand binding and the level of expression were studied. The expression of the serotonin transporter in tunicamycin-treated Sf9 cells resulted in low levels of ligand binding activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated serotonin transporters that contained reduced numbers of N-linked glycosylation sites had unchanged Kd for [125I]RT155 binding whether there were 2, 1, or 0 N-linked glycosylation sites present on the serotonin transporter. In contrast, Bmax was dramatically reduced; levels of expression of the unglycosylated serotonin transporter (0.4 pmol/mg) were 20-fold lower compared with levels of the fully glycosylated serotonin transporter. The Km for [3H]serotonin uptake was also unchanged. These data indicate that glycosylation is required for optimal stability of the serotonin transporter in the membrane but not for serotonin transport or ligand binding per se.
Using whole viable human colon carcinoma HT29 cells as immunogen, we produced a monoclonal antibody (mAb) termed 69-6-5. The antibody was functionally selected on its anti-cell-spreading activity. By immunoprecipitation of surface radiolabeled cell lysates from HT29-D4 cells (an HT29 cell clone), mAb 69-6-5 recognized a molecular complex resembling integrin heterodimers. Sequential immunodepletions with mAb to the integrin alpha v subunit demonstrated that this complex was composed of alpha v-containing integrins. Accordingly, mAb 69-6-5 reacted with integrin alpha v beta 3 immunopurified from melanoma cells and integrins alpha v beta 5 and alpha v beta 6 immunopurified from pancreatic carcinoma cells. In cell adhesion assays, the 69-6-5 mAb was able to inhibit strongly in a dose-dependent manner arginine-glycine-aspartic acid-mediated adhesion of HT29-D4 cells to vitronectin, fibronectin, or ProNectin F but not to laminin or collagen. Immunoprecipitations with beta chain-specific antisera indicated that these cells express integrins alpha v beta 5 (receptor for vitronectin) and alpha v beta 6 (receptor for fibronectin) but neither alpha v beta 1 nor alpha v beta 3. In summary, these results indicated that mAb 69-6-5 reacts with several alpha v integrins and that it can effectively interfere with the adhesive functions of at least alpha v beta 5 and alpha v beta 6, which represent the major receptors on HT29-D4 cells responsible for their adhesion on vitronectin and fibronectin.
Antigenic determinants recognized by human proinsulin (HPI)-specific monoclonal antibodies (Mabs) and Mabs crossreacting with free human C-peptide (HCP) were mapped by using various forms of purified, partially converted HPI intermediates. Two HPI-specific mouse Mabs (GS-4G9 and GS-9A8) reacted with the same antigenic determinant, GS, which was localized to the site of linkage of the B-chain to the C-peptide (Arg-Arg) at positions 31-32. These antibodies bind with equal efficiency to C65-A1 split proinsulin and to intact HPI. The binding of C32-C33 split proinsulin is markedly reduced. A rat Mab (GN-VIIB6), which crossreacts with free HCP in addition to HPI, reacted similarly with various HPI intermediates as it had with the corresponding synthetic HCP fragments, as previously reported (see ref. 9). This determinant (GN) is a three-dimensional structure composed of residues located in two separate regions in the C-peptide segment (positions 40-45 and 57-63). Reduced, carboxymethylated HPI retains the GN-determinant, whereas all insulin-like immunoreactivity identified with a conventional guinea pig insulin antiserum is completely lost. The binding of the two GS Mabs to the denatured HPI was reduced by 40-50% compared with intact HPI. It is concluded that the strong GN-determinant can readily form in the C-peptide segment of HPI, independently of the presence of ordered structure in the insulin moiety. A predicted beta-turn at position 47-50 may play an important role in bringing N- and C-terminal regions of the C-peptide segment into close proximity.(ABSTRACT TRUNCATED AT 250 WORDS)
An antigenic determinant present in the cytoplasm, but not on the surface membrane of human Ia+ cells, is defined by a monoclonal antibody (VIC-Y1) and is shown by immunoprecipitation and by NEPHGE to be expressed by Ia oligomers. Immunoprecipitations of cellfree translates and of purified Ia subunits indicate that the VIC-Y1 determinant is located on the Ia gamma-(invariant) chain, as well as on two other related molecules, provisionally termed gamma 2 and gamma 3. Within our experimental conditions, the three forms of gamma-chains co-precipitate exclusively with Ia oligomers. As detected by VIC-Y1 and in the limits of our assays, gamma-chains could not be found at the cell surface; their tissue distribution, determined by cytoplasmic indirect immunofluorescence with VIC-Y1, closely resembles that of Ia antigens, with the possible exception of acute lymphatic leukemia cells (Ia+, gamma-chain-).
The development of a simple microscale solid phase screening RIA and improved methods for cell cloning has lead to the establishment of 2 prohormone- and species-specific mouse monoclonal antibodies against human proinsulin (HPI). These antibodies react to determinant(s) only expressed on the HPI molecule. In addition, 11 rat monoclonal antibodies were generated which react with both human C-peptide (HCP) and HPI. All 11 rat antibodies recognize a very similar antigenic determinant in the C-peptide that appears to be made up of residues 40-45 and 57-63, which are probably brought into close proximity by a beta-turn near the center of the connecting segment. The identical behavior of both HPI-specific mouse antibodies in competition experiments indicates that the antigenic structure recognized in proinsulin might be the same for both antibodies. This structure could not be regenerated by mixing equimolar amounts of human insulin and C-peptide, including the chemically synthesized complete proinsulin connecting segment (Arg X Arg X HCP X Lys X Arg), which contains the entire sequence removed from proinsulin in the conversion to mature insulin. Indirect evidence is provided that a HPI molecule simultaneously can bind a C-peptide-directed rat antibody and a HPI-specific mouse antibody when the first antibody is presented to HPI in solution phase. However, when HPI is immobilized on a plastic surface, the binding of 1 type of antibody completely blocks the binding of the other. These antibodies provide useful new tools for studying the biosynthesis and 3-dimensional structure of HPI and HCP.
Monoclonal antibodies (McAbs) against the myosin heavy chain (MHC) of adult chicken pectoralis muscle have been tested for reactivity with pectoralis myosin at selected stages of chick development in vivo and in vitro. Three such McAbs, MF 20 and MF 14, which bind to light meromyosin, and MF 30, which binds to myosin subfragment two (S2), were used to assay the appearance and accumulation of specific MHC epitopes with: (a) indirect, solid phase radioimmune assay (RIA), (b) immunoautoradiography, (c) immunofluorescence microscopy. McAb MF 20 bound strongly and equivalently to MHC at all stages of embryonic development in vivo. In contrast, the MF 30 epitope was barely detectable at 12 d of incubation but its concentration rose rapidly just before hatching. No detectable binding of MF 14 to pectoralis myosin could be measured during myogenesis in vivo until 1 wk after hatching. Immunofluorescence studies revealed that all three epitopes accumulate in the same myocytes of the developing pectoralis muscle. Since all three McAbs bound with high activity to native and denatured forms of myosin, it is unlikely that differential antibody reactivity can be explained by conformational changes in myosin during development in vivo. When myogenesis in vitro was monitored using the same McAbs, MF 20 bound to the MHC at all stages tested while reactivity of MF 30 and MF 14 with myosin from cultured muscle was never observed. Thus, this study demonstrates three different immunochemical states of the MHC during development in vivo of chick pectoralis muscle and the absence of later occurring immunochemical transitions in the MHC of cultured embryonic muscle.
We report a recurrent idiotype on a remarkably high fraction (4/19) of murine monoclonal antibodies specific for human Ia monomorphic determinants and elicited by separate immunizations. For three of them, the shared idiotype is associated with the antigen-combining site. These results indicate that the spectrum of mouse antibody responses to human Ia antigens may be based on recurrent idiotypes, suggesting a limited potential repertoire of murine monoclonal antibodies to human Ia antigens. Anti-idiotypic reagents might be helpful in dissecting this repertoire and to generate a mirror image of a human Ia antigenic map. Furthermore, antisera to the idiotype of antibodies specific for human Ia monomorphic determinants might help in elucidating the interactions between Ia molecules and receptors on immune cells.
A combination of serological (cytotoxicity, binding assay, lysostrip) and immunochemical (indirect immunoprecipitation, sequential immunoprecipitation, two-dimensional gel electrophoresis) assays have shown that the monoclonal antibodies Q6/64 recognizes an antigenic determinant which is expressed by certain gene products of the A and b loci of the HLA region. The determinant identified by Q6/64 is spatially close to those which define the serological polymorphism of the HLA-A, B, C antigenic system. The results presented in this study in conjunction which those recently published by other investigators indicate that sharing of determinants among HLA-A and -B allospecificities is more frequent than originally assumed on the basis of the cross-reactivity pattern obtained with alloantisera. This conclusion is in agreement with the high degree of homology in the primary amino-acid sequence of A and B allospecificities.
The effect of glycosylation on the assembly and antigenicity of HLA antigens was investigated by examining HLA antigens synthesized in the presence of the antibiotic tunicamycin, an inhibitor of asparagine-linked oligosaccharide addition, with monoclonal antibodies specific for a variety of antigenic determinants. The monoclonal antibody Q5/13 reactive with a determinant expressed on the beta chain of human Ia-like antigens immunoprecipitated alpha and beta subunits with reduced apparent molecular weights from tunicamycin-treated cells, indicating that glycosylation is not required for association of the Ia-like antigen alpha and beta subunits. Immunoprecipitation of HLA-A,B,C antigens from tunicamycin-treated cells with four monoclonal antibodies specific for the heavy chain and one specific for beta 2-microglobulin showed that the heavy-chain determinant detected by the antibody Q6/64 is absent from the non-glycosylated molecule. This is the first demonstration that carbohydrate addition during biosynthesis affects the protein conformation of the HLA-A,B,C heavy chain.