Murine Peptide Research Articles

Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor.

Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue. The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel. The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10. Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel. However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel. These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF. It appears that the epitope is generated by a posttranslational modification of the peptide.

Crossrecognition by CD8 T cell receptor alpha beta cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp60 which share intermediate sequence homology.

Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H-2Db)-restricted CD8 T cell receptor (TcR) alpha/beta cells. A CD8 TcR alpha/beta clone with specificity for the mycobacterial hsp60 peptide499-508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon-gamma (IFN-gamma) stimulation. The stress-induced, self peptide could be extracted from IFN-gamma-stressed macrophages by acid elution, suggesting that the IFN-gamma-induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60-specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9-mer to 11-mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H-2Db binding were tested in CTL assays. The overlapping murine hsp60 peptides162-170/171 were stimulatory at a concentration as low as 10-100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10-100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN-gamma-stressed macrophages to the cross-reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.

Major differences in transporter associated with antigen presentation (TAP)-dependent translocation of MHC class I-presentable peptides and the effect of flanking sequences.

The MHC-encoded transporter associated with Ag presentation (TAP) translocates peptides from the cytosol to the ER lumen, where association with MHC class I molecules occurs. The MHC class I/peptide complex is subsequently transported to the cell surface for presentation to CD8+T cells. We studied TAP-dependent translocation of defined MHC class I presentable murine peptides by competition for translocation of a radiolabeled model peptide, to address whether efficient peptide presentation by MHC class I molecules is preceded by equal efficient peptide translocation by TAP. Surprisingly, we observed that four immunodominant viral peptides of 16 peptides tested were very inefficiently transported by TAP. Inefficient translocation could be overcome by substitution of a proline residue present at position 3 in the peptides. Furthermore, addition of natural flanking amino acids directly surrounding a poorly transported peptide could considerably improve translocation by TAP. Our data suggest that some peptides are efficiently transported by TAP in their optimal size for MHC class I binding, whereas other peptides are transported as larger peptide fragments that need further trimming in the ER for MHC class I binding.

Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus.

Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (M(r) = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-r in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used.(ABSTRACT TRUNCATED AT 400 WORDS)

Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8+ tumor-infiltrating lymphocytes.

The B16/BL6 melanoma is a relatively nonimmunogenic tumor expressing low levels of MHC class I molecules. BL6 clones expressing transfected H-2Kb class I molecules were, by contrast, highly immunogenic in immunocompetent mice. Tumor-infiltrating lymphocytes (TILs) generated from the H-2Kb+ BL6 lesions (Thy 1.2+, CD8+, CD4-) efficiently lysed H-2Kb+ melanoma (CL8-1 and 2Kb38) and the H-2Kb+ nonrelated 3-methylcholanthrene (MCA)-105 sarcoma, but not the H-2Kb- parental melanoma BL6-8. This strongly suggests that CL8-1, 2Kb38, and MCA-105 express identical or cross-reactive T cell epitopes recognized by CL8-1 TILs in the context of the H-2Kb class I allele. To identify the T cell epitopes, peptides were acid-eluted from various cells, and fractionated by HPLC. Five HPLC fractions (F1mel-F5mel) of 70 tested contained peptides derived from H-2Kb+ CL8-1 melanoma (but not H-2Kb- melanomas) that were capable of conferring susceptibility to CL8-1 TIL lysis on H-2b-expressing target cells (EL4, C1R.Kb), but not on H-2d-expressing P815 target cells. CL8-1 TILs failed to recognize peptides derived from H-2Kb+ nonmelanoma targets such as EL4 or normal B6 splenocytes. Interestingly, CL8-1 TILs appeared to recognize peptide species contained in two HPLC fractions derived from the MCA-105 sarcoma (F1sar and F5sar). Conversely, TILs derived from MCA-105 lesions recognized MCA-105 and CL8-1 tumor cells, as well as F5mel and F5sar peptides presented by EL4 targets. These data support common murine tumor-associated peptide epitopes presented by H-2Kb and recognized by CD8+ CTLs derived from histologically distinct tumors, melanoma and sarcoma.

Inhibition of human and murine cytosolic epoxide hydrolase by group-selective reagents

1. 1. Human and murine cytosolic epoxide hydrolase were inhibited by thiol-, imidazole- and carboxyl-selective reagents. They were not inhibited by amino-, guanido- or activated serine-selective reagents. 2. 2. Murine, but not human, cytosolic epoxide hydrolase was inhibited by N-bromosuccinimide, a tryptophan selective reagent. 3. 3. Based on sequence data from peptides isolated from CNBr digests, human and murine CEH share areas of sequence homology. Of the five unique human CEH CNBr peptides sequenced, three shared common sequences with one of the unique murine CEH CNBr peptides. The human and murine CEH peptides with common sequences had between 64 and 78% sequence identity. 4. 4. A cysteine important for the activity of murine CEH appears not to be in the active site as judged by N-phenylmaleimide inhibition in the presence and absence of either (2S,3S)-2,3-epoxy-3-(4-nitrophenyl)glycidol, a competitive inhibitor, or trans-stilbene oxide, a substrate.

Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice

Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103–116 YKTTICGKGLSATV and 139–151 HCLGKWLGHPDKF are encephalitogenic determinants in SWR/J (H-2 q) and SJL/J (H-2 s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In SWR/J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105–115 TTICGKGLSAT and PLP 106–116 TICGKGLSATV are encephalitogenic in SWR/J mice, but PLP 106–115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortes PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141–149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.

Production and characterization of two new mouse monoclonal antibodies reactive with denatured mouse class II beta chains.

We report on the production and characterization of two murine peptide specific anti-major histocompatibility complex class II chain specific monoclonal antibodies. Two new mouse monoclonal antibodies reactive with two synthetic peptides corresponding to Abb amino acids (146-157) and Abs amino acids (146-157) were produced. KL295 is the mouse anti-Abb monoclonal antibody, which reacts with denatured beta chains of H-2b, H-2d, H-2p, and H-2q, but fail to react with spleen cell lysates from H-2f, H-2j, H-2k and H-2s mice. The mouse anti Abs monoclonal antibody, KL304 in contrast reacts with the denatured Class II beta chains of H-2f, H-2j, H-2k and H-2s mice, but fails to bind H-2b, H-2d, H-2p or H-2q beta chain, suggesting residues 153-155 of this molecule to be critical for the epitope. Both KL295 and KL304 bind B10.WB (H-2j) which suggests a unique epitope in this strain of mice. Neither KL295 or KL304 react with native mouse class II cell surface molecules.

Experimental myasthenia gravis in congenic mice. Sequence mapping and H‐2 restriction of T helper epitopes on the α subunits of Torpedo californica and murine acetylcholine receptors

Immunization of mice with nicotinic acetylcholine receptor from Torpedo electric organ (TAChR) causes a disease similar to human myasthenia gravis (experimental autoimmune myasthenia gravis, EAMG). Susceptibility to EAMG correlates with the H-2 haplotype. In this study we used overlapping synthetic peptide corresponding to the complete sequences of the alpha subunits from TAChR and murine muscle AChR (MAChR) to map T helper epitopes in congenic murine strains of different H-2 haplotype. C57BL/6 and BALB/B mice (highly susceptible to EAMG) and BALB/c and CB17 mice (less susceptible to EAMG), immunized with TAChR, developed similar anti-TAChR antibody titers and L3T4+ (T helper) cell sensitization. Different sequence segments of the TAChR alpha subunit were recognized by L3T4+ cells from strains of H-2b and H-2d haplotype. The sequence segments recognized by the H-2d strains have the highest predicted propensity to form amphipatic alpha helices, while those recognized by the H-2b strains do not. We investigated whether in EAMG T helper cells cross-react with autologous AChR sequences, and a true break of the tolerance occurs. Overlapping synthetic peptides, corresponding to the complete sequence of MAChR alpha subunit, were used to test L3T4+ cell from mice immunized with TAChR. L3T4+ cell strains did not cross-react with any murine peptide sequence, while L3T4+ cells from H-2d mice were strongly stimulated by the peptide sequence Ma alpha 304-322, which is very similar to the homologous Torpedo peptide.

The Q7 alpha 3 domain alters T cell recognition of class I antigens.

In this study we have analyzed the role of the alpha 3 domain of class I molecules in T cell recognition. Using the laboratory engineered molecules LLQQ (alpha 1/alpha 2 from Ld, alpha 3, and phosphatidyl inositol (PI) linked C terminus from Q7) and LLQL (alpha 1/alpha 2 from Ld, alpha 3 from Q7, transmembrane (TM) and cytoplasmic domains from Ld) we show that these molecules are not recognized by primary Ld-specific CTL. The cell membrane expression of both Ld and LLQL are upregulated by co-culture with an exogenously supplied murine cytomegalovirus-derived peptide indicating that the Q7 alpha 3 domain does not interfere with binding of Ag to alpha 1/alpha 2. However, only peptide pulsed Ld but not LLQL target cells are recognized by Ld-restricted-peptide specific CTL. In contrast to the above results, LLQL and LLQQ molecules can be recognized by bulk alloreactive anti-Ld CTL and 2/3 of CTL clones derived from in vivo primed mice. The fact that these secondary CTL recognize LLQQ indicates that a PI linkage is permissive for presentation of class I epitopes to alloreactive CTL. These secondary CTL are resistant to blocking at the effector stage by mAb against CD8 and express relatively low levels of membrane CD8 molecules compared to CTL from unprimed mice. Further, culture of unprimed CTL precursors in the presence of CD8 mAb also allows for the generation of CD8-independent CTL that recognize LLQL. Taken together, these data indicate that the alpha 3 domain of Q7 (Qa-2) prevents CD8-dependent CTL from recognizing Ld, regardless of whether the class I molecule is attached to the cell surface by a PI moiety or as a membrane spanning protein domain. We hypothesize that this defect in recognition is most likely due to an inability of CD8 to interact efficiently with the Q7 alpha 3 domain and could account for why Q7 molecules do not serve as restricting elements for virus and minor H-Ag-specific CTL.

Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells.

Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.