Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain

6 pages, 3 figures, 2 tables.-- PMID: 15901806 [PubMed].-- Printed version published Aug 2005.Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([3H]MEGY). In addition, we have also synthesized two analogs: (D-Ala2)-MEGY (Y-D-Ala-GFMGY) and (D-Ala2, Val5)-MEGY (Y-D-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [3H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a D-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [3H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [3H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.This study was supported by Ministerio de Educación y Ciencia (SAF2004-05144), Junta de Castilla y León (SA031/03/00B), and Hungarian National Scientific Research Foundation (Hungary) Grant T046514Peer reviewe

Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes.

Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition.

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2Db using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind Db. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to Db. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the Db pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited Db-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all Db-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, Db-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to Db, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule

Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand