Biomedical subjects

D F Hunt

Publications and source records attributed to D F Hunt.

At least 55 records · Page 3Linked to original sources

PubMed1997

A naturally processed peptide presented by HLA-A*0201 is expressed at low abundance and recognized by an alloreactive CD8+ cytotoxic T cell with apparent high affinity.

In contrast to T cells that respond to peptides presented by self MHC molecules, alloreactive T cells recognize determinants expressed on nonself MHC molecules. Because current positive selection models suggest that T cell affinity toward a nonself MHC molecule would be lower than that toward a self MHC molecule, we previously proposed that vigorous alloreactive responses would be generated preferentially toward those antigenic peptide complexes presented at the highest density on the cell surface. The high abundance of two class I MHC-associated peptides that have been identified as allo- or xenoantigens is consistent with this hypothesis. We report here the identification of a naturally processed peptide YLDPAQQNL that is presented by HLA-A*0201 and recognized by an alloreactive T cell clone. This peptide appears to originate from an unknown member of the zinc finger proteins. Quantitation by mass spectrometry indicates that this peptide is present on the surface at 85 to 125 copies per cell, comparable with the density of several other epitopes presented by HLA-A*0201 to self MHC-restricted T cells. In addition, based on the affinity of the peptide for HLA-A*0201 and the half-maximal peptide concentration required for T cell sensitization, this alloreactive T cell appears to have an affinity similar to or higher than that of many self MHC-restricted T cells. These data suggest that allogeneic responses can be directed against antigenic determinants of low abundance and that recognition of alloreactive peptides is not limited by a lower affinity of T cells for nonself MHC molecules.

Clone Cells↗

PubMed1997

Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Actin Cytoskeleton↗

PubMed1997

The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

Amino Acid Sequence↗

PubMed1997

HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides.

Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

Amino Acid Sequence↗

PubMed1997

Susceptibility to ankylosing spondylitis correlates with the C-terminal residue of peptides presented by various HLA-B27 subtypes.

Susceptibility to spondyloarthropaties is strongly associated with some HLA-B27 alleles. Evidence suggests a direct pathogenic role for the B27 molecules which possibly present an arthritogenic peptide to the T cells. If this hypothesis is true, B27 subtypes that differ structurally but are disease-associated ought to be capable of presenting such peptide(s), while non-disease-associated ones would not. We have recently described a B27 subtype, B*2709, and shown its absence in ankylosing spondylitis (AS) patients. Here, we show the elution and sequence of peptides from HLA-B*2709 molecules. Similar to other B27 subtypes, these peptides are mainly nonamers with an Arg at position P2. Comparison of the C-terminal anchors of peptides eluted from B*2702 and B*2705 with those eluted from B*2709 reveals that, while B*2702 and B*2705 have a broader specificity, B*2709 molecules appear to only accept C-terminal hydrophobic residues. A common feature shared by the two caucasoid AS-associated subtypes (B*2702 and B*2705) but different from B*2709, is the presence of a Tyr as peptide C-terminal anchor. The substitution of Val for Tyr at the C terminus in one of the eluted peptides greatly reduces the binding to B*2709 molecules. This finding suggests Tyr as a discriminative amino acid allowed at the C terminus of peptides bound to the AS-associated B27 subtypes, but not to those which are not associated with AS.

Amino Acid Sequence↗

PubMed1997

Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B.

Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.

Amino Acid Sequence↗

PubMed1997

The isolation of parvalbumin isoforms from the tail muscle of the American alligator (Alligator mississipiensis).

Multiple parvalbumin isoforms have been detected in the tail (skeletal) muscle of the American alligator (Alligator mississipiensis). One of these isoforms (APV-1) has been highly purified and partially characterized. Protein purification involved mainly gel filtration and anion exchange chromatography, and characterization included gel electrophoresis, amino acid composition analysis, metal ion analysis, MALDI-TOF and ESI mass spectrometry, ultraviolet and fluorescence spectroscopy, and one- and two-dimensional 500 MHz proton NMR spectroscopy. The alligator isoforms are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins. In fact, the one highly purified isoform that forms the basis of this study has only phenyl-alanine as an aromatic residue. Ion exchange chromatography further indicates that this isoform has a relatively high isoelectric point (pl approximately 5.0), indicating that it is an alpha-lineage parvalbumin. This alligator parvalbumin isoform is unusual in that it has an atypically high Ca2+ content (almost 3.0 mole of Ca2+ per mole of protein) following purification, a fact supported by terbium fluorescence titration experiments. Preliminary comparative analysis of the highly purified alligator parvalbumin isoform (in the Ca2-loaded state) by two-dimensional 1H-NMR (2D 1H TOCSY and 2D 1H NOESY) indicates that there is considerable similarity in structure between the alligator protein and a homologous protein obtained from the silver hake (a saltwater fish species).

Alligators and Crocodiles↗

PubMed1997

The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition.

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Animals↗

PubMed1997

Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes.

To understand how thymic selection gives rise to T cells that are capable of major histocompatibility complex (MHC)-restricted recognition of antigen but are tolerant of self, we directly examined how peptide/MHC ligands expressed on thymic epithelial cells trigger the positive selection of immature thymocytes. We demonstrate that abundant self-peptides, purified from the H-2D(b) molecules of thymic epithelial cells, are specifically recognized during the positive selection of CD8+ T cells, implying that positive selection generates a repertoire of T cells that is weakly self-reactive. We also found that this recognition is somewhat cross-reactive, thereby providing an explanation for how the specific recognition of a limited repertoire of thymic self-peptides can select a diverse repertoire of T cells.

Animals↗

PubMed1997

Humoral immune responses to cathepsin D and glucose-regulated protein 78 in ovarian cancer patients.

Many cancer patients develop tumor-reactive immune responses against antigens that are either expressed on the surface of tumor cells or released from them into the peripheral circulation. In this study, tumor-reactive immunoglobulins, present in the sera of ovarian cancer patients, were used to identify commonly recognized tumor-associated antigens on ovarian tumor cells. Western immunoblot analysis of cellular proteins, obtained from UL-1 ovarian tumor cell line, demonstrated several commonly recognized immunoreactive proteins. Two of these proteins (Mr 32,000 and 71,000) were selected for further investigation. Cellular proteins isolated from normal human ovarian epithelia, in a similar fashion, failed to exhibit corresponding immunoreactivity to these proteins. As an additional control, sera from normal (nontumor-bearing) individuals failed to identify these proteins on Western immunoblots. Furthermore, the absorption of the ovarian cancer patients' sera with normal ovarian epithelial tissue did not remove the reactivity of these two proteins. The Mr 32,000 and 71,000 proteins were subsequently purified by reverse-phase high-performance liquid chromatography, separated by SDS-PAGE, transferred to the polyvinylidene difluoride membrane, and digested with trypsin. These resulting tryptic fragments were separated by microbore reverse-phase high-performance liquid chromatography, and selected fragments were sequenced by mass spectrometry. This sequence analysis identified the Mr 32,000 protein as cathepsin D and the Mr 71,000 as glucose-regulated protein 78 (member of the heat shock protein family). The identities of cathepsin D and glucose-regulated protein 78 were confirmed by Western blot analysis. Additionally, the presence of cathepsin D was demonstrated in association with immune complexes in vivo. Currently, the common antigenic epitopes of these proteins are being defined.

Adenocarcinoma, Mucinous↗

PubMed1996

Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100.

Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.

Amino Acid Sequence↗

PubMed1996

Protamines of reptiles.

We have characterized for the first time the complete primary structure of the main protamine components of the sperm from four reptiles: Chrysemys picta (turtle), Elaphe obsoleta (snake), Anolis carolinensis (lizard), and Alligator mississipiensis (crocodilian). These species were chosen to represent one of each of the main phylogenetic branches of this taxonomic group. Comparison of these protamine sequences with those already available from other vertebrate groups allows us to define properly the chemical consensus composition of protamines and provides a unique insight into their molecular evolution and classification.

Alligators and Crocodiles↗

PubMed1996

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Amino Acid Sequence↗

PubMed1996

G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells.

The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.

Amino Acid Sequence↗

PubMed1996

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

Amino Acid Sequence↗

PubMed1996

Isolation of NEB-LFamide, a novel myotropic neuropeptide from the grey fleshfly.

A methanolic extract of 350,000 adult grey fleshflies Neobellieria bullata, was prepared and screened for myotropic activity. After fractionation on the first column, all fractions were screened in two heterologous (Locusta oviduct and Leucophaea hindgut) and one homologous (Neobellieria hindgut) myotropic bioassay. We here report the purification of one fraction, which stimulates the contractions of the Locusta oviduct. Electrospray Mass Spectrometry of the peptide revealed a molecular mass of 1395.82. The primary structure has been determined as AYRKPPFNGSLF-amide. This novel peptide was designated Neb-LF-amide. This sequence is different from the other known myotropic peptides in insects. The threshold concentration of the synthetic peptide is 1 x 10(-7) M on the Locusta oviduct. On the hindgut of Neobellieria or Leucophaea, the synthetic peptide is not active. By use of a polyclonal antiserum raised against the synthetic peptide, immunoreactivity was localized in median neurosecretory cells in the pars intercerebralis of the fly brain, indicating that Neb-LF-amide is a neuropeptide.

Amino Acid Sequence↗

PubMed1996

Mimosine targets serine hydroxymethyltransferase.

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G1/S boundary, and blocks DNA replication when added to S phase cells after a lag of approximately 2.5 h. We have shown previously that [3H]mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [3H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly- cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly- cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.

Amino Acid Sequence↗

PubMed1996

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.

Amino Acid Sequence↗