A novel active pentapeptide from chicken brain identified by antibodies to FMRFamide

The tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and peptides structurally related to it, have been isolated from molluscan ganglia. They have widespread actions on both invertebrate and vertebrate tissues and there is increasing evidence that they are an important group of invertebrate peptide neurotransmitters. It is of interest that the primary amino acid sequence of FMRFamide forms the C-terminal tetrapeptide of an enkephalin-like heptapeptide (Met-enkephalin-ArgPhe) isolated from bovine adrenal medulla and striatum. Antisera to FMRFamide have been shown to react in radioimmunoassay and immunohistochemistry with material in the central nervous system of various vertebrate species, but the identity of this material, and in particular its relationship to the opioid heptapeptide, remains uncertain. We have used antibodies specific for the C-terminus of FMRFamide in radioimmunoassays to monitor purification of the material in chicken brain. We describe here the sequence of one of the peptides obtained. It is a biologically active peptide which does not seem to be related to other known vertebrate neuropeptides.     

The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region

The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.     

Structure of a genomic clone encoding biologically active human relaxin

Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.     

Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides

Naturally occurring peptides with biological actions have in most cases been detected by observing their biological activities in crude extracts and their isolation has been followed using bioassays. As a complement to the classical biological detection systems, we have proposed a chemical detection system based on fragmentation of peptides in tissue extracts followed by identification of certain of these peptide fragments having distinct chemical features. One such chemical feature is the C-terminal amide structure which is characteristic of many biologically active peptides. We have devised a chemical assay method for peptides having such a structure and have found several previously unknown peptide amides in procine upper small intestinal tissues. We report here the isolation and characterization of two of them, designated PHI and PYY. PHI is related to secretin, vasoactive intestinal polypeptide (VIP, glucagon and gastric inhibitory polypeptide (GIP); PYY is related to the pancreatic polypeptide and to neurotensin. Both peptides exhibit biological activities and appear to be present not only in the intestine but also in brain.     

Polymorphism and absence of Leu-enkephalin sequences in proenkephalin genes in Xenopus laevis

The structures of the genes coding for the opioid peptide precursors proopiomelanocortin, proenkephalin (proenkephalin A) and prodynorphin (proenkephalin B), are known for some mammalian species. To gain insight into the evolutionary history of these precursors, we have examined the proenkephalin gene in the South African clawed toad, Xenopus laevis, which diverged from the principal line of vertebrate evolution some 350 Myr ago. The human proenkephalin gene consists of four exons, of which the main exon (exon IV) contains all known biologically active peptides--six Met-enkephalin sequences and one Leu-enkephalin sequence. We report here the primary structures of the putative main exons of two proenkephalin genes in X. laevis, each of which codes for seven Met-enkephalin sequences but no Leu-enkephalin, indicating that Met-enkephalin preceded Leu-enkephalin in the evolution of the proenkephalin gene. The organization of the main exons of the toad genes is remarkably similar to that of the human gene and conserved regions provide evidence for functionally significant structures. We also detect a polymorphism in one of the toad proenkephalin genes, mapping 1.5 kilobases (kb) 5' of the main exon; it is caused by an insertion/deletion of a 1-kb repetitive sequence which has the characteristics of a transposable element.     

Immunoglobulin-like nature of the alpha-chain of a human T-cell antigen/MHC receptor

Although the receptor with which T cells bind specific antigen can, like immunoglobulin, distinguish between antigens which differ only slightly in structure, it is unique in recognizing antigen only in conjunction with one of the self proteins of the major histocompatibility complex (MHC restriction). The receptor was identified and characterized in mouse and man by using monoclonal antibodies to receptor idiotypes, and consists of two disulphide-linked polypeptides, and acidic alpha-chain and a neutral to slightly basic beta-chain. Peptide maps have shown that, like immunoglobulin, both chains vary for receptors of different specificities. T-cell-derived cDNA clones have recently been identified in mouse and man encoding immunoglobulin-like molecules. These were identified as derived from beta-chain genes through a partial N-terminal protein sequence of the beta-chain isolated from a human T-cell tumour. We have now purified the alpha- and beta-chains of the receptor of the human T-cell leukaemia line HPB-MLT, and have determined the amino acid sequence of several tryptic peptides derived from each chain. Our results further confirm that the previously reported cDNA clones encode beta-chains. The sequence of the alpha-chain peptides identify this as another immunoglobulin-like polypeptide chain. Particularly striking was an alpha-chain peptide with high homology to the conserved portion of the immunoglobulin J segment and T-cell receptor beta-chains. Surprisingly, the alpha-chain peptides show little similarity to the sequence predicted by two overlapping putative murine alpha-chain cDNA clones.     

Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T-cell determinants

Study of the immune response to synthetic antigens has shown that uncoupled peptides can realize their potential as vaccines only if they contain domains that react with helper T-cell receptors and Ia antigens in addition to antibody binding sites. Here we consider whether genetically restricted non-responsiveness to an uncoupled peptide could be overcome by synthesizing a peptide with an additional helper T-cell epitope from a different protein. We demonstrate that H-2d mice, which are non-responders to the 141-160 VP1 peptide of foot-and-mouth disease virus (FMDV), can be converted into responders by immunization with peptides containing the FMDV sequence with defined 'foreign' helper T-cell determinants from ovalbumin or sperm whale myoglobin. Furthermore, the virus-neutralizing activity of the antibody raised against peptide was dependent on the determinant used. Thus, FMDV peptides with the added sequences 323-339 from ovalbumin and 132-148 from sperm-whale myoglobin elicited a high degree of neutralizing activity in B10.D2 mice. The sera from mice which received the peptide with the added sequence 105-121 from sperm whale myoglobin did not neutralize the virus, although they had high levels of anti-141-160 FMDV peptide activity. Our data indicate that the T-cell help given by the 'foreign' epitopes is B-cell clone specific. These results are likely to have important implications for the design of peptide vaccines.     

Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein

Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant. The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.     

Antisera to a synthetic peptide of the sis viral oncogene product recognize human platelet-derived growth factor

It has recently been reported that the sequences of the sis oncogene of simian sarcoma virus (SSV) and of human platelet-derived growth factor (PDGF) are very similar, establishing the most solid link yet between the mitogenic actions of growth factors and the transforming proteins of retroviruses. To investigate molecular mechanisms of transformation I have produced antisera against synthetic peptides corresponding to segments of the protein sequences predicted by the nucleotide sequences of viral oncogenes. Applying this approach to the case of sis and PDGF, I report here the results of probing outdated human platelets with an antiserum directed against a synthetic peptide representing residues 139-155 of the predicted sequence of the SSV transforming protein, p28sis (ref. 3). I detected peptides of apparent molecular weights (MWs) 30,000 to 31,000 (30-31K) and 16-18K, which correspond to the apparent molecular weights of nonreduced and reduced PDGF. In addition, a peptide of MW 21,000 was detected in platelets and a protein of MW 56,000 was detected in SSV-infected marmoset cells.     

Homology of proteasome subunits to a major histocompatibility complex-linked LMP gene.

The class II region of the major histocompatibility complex (MHC) contains genes encoding at least two subunits of a large, intracellular protein complex (the low molecular mass polypeptide, or LMP, complex). This complex is biochemically similar to the proteasome, an abundant and well conserved protein complex having multiple proteolytic activities. Here we report the isolation of a complementary DNA corresponding to one of the subunits of the LMP complex, LMP-2. The protein predicted from this cDNA sequence closely matches the amino-terminal peptide sequence of a rat proteasome subunit, confirming that the proteasome and the LMP complex share polypeptide subunits. The LMP-2 gene is tightly linked to HAM1, a gene thought to be required for translocating peptide fragments of endogenous antigens into the endoplasmic reticulum for association with MHC class I molecules. These observations suggest that the LMP complex may be responsible for generating peptides from cytoplasmic antigen during antigen processing.     

Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.     

A dynorphinergic pathway of Leu-enkephalin production in rat substantia nigra

The amino acid sequence of the opioid peptide Leu-enkephalin is found within several larger peptides, which are generated from the precursors proenkephalin and prodynorphin. Proenkephalin contains four copies of the sequence of Met-enkephalin, a single copy of the sequence of Leu-enkephalin and one copy each of two extended Met-enkephalin sequences. Proenkephalin contains three peptides--alpha-neo-endorphin, dynorphin A and dynorphin B--the N-terminal sequences of which are identical to that of Leu-enkephalin. There is good evidence that the large amounts of Leu-enkephalin found in the adrenal medulla are generated from the precursor proenkephalin, but as yet prodynorphin has not been shown to be processed to yield Leu-enkephalin. We show here that the relatively high levels of Leu-enkephalin found in the rat substantia nigra are supplied by striatonigral axons and generated from the precursor prodynorphin.     

Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide

Proteins secreted by prokaryotic cells are synthesized as precursors containing an amino-terminal extension sequence or signal peptide. Although these signal peptides share little primary sequence homology, recent studies suggest that they function via common pathways during the transport process and that a common element may reside in their secondary structural characteristics. We are investigating the role of an idealized hydrophobic sequence with high potential for alpha-helix formation in the Escherichia coli alkaline phosphatase signal peptide. Here, amino-acid substitutions were made using site-directed mutagenesis to produce a mutant signal sequence containing nine consecutive leucine residues in the hydrophobic core segment. Transport studies with this mutant precursor indicate that mature alkaline phosphatase is correctly targeted to the E. coli periplasm and that processing of the precursor to the mature form of the enzyme is extremely rapid. In contrast, processing is slowed when the mutant signal sequence is lengthened by the insertion of five additional leucine residues and one serine.     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs.

T cells recognize foreign protein antigens in the form of peptide fragments bound tightly to the outer aspect of molecules encoded by the major histocompatibility complex (MHC). Most of the amino-acid differences that distinguish MHC allelic variants line the peptide-binding cleft, and different allelic forms of MHC molecules bind distinct peptides. It has been demonstrated that peptide-binding to MHC class I involves anchor residues in certain positions and that antigenic peptides associated with MHC class I exhibit allele-specific structural motifs. We have previously reported an analysis of MHC class II-associated peptide sequences. Here we extend this analysis and show that certain amino-acid residues occur at particular positions in the sequence of peptides binding to a given MHC class II molecule. These sequence motifs require the amino terminus to be shifted one or two positions to obtain alignment; such shifts occur naturally for a single peptide sequence without qualitatively altering CD4 T-cell recognition.     

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.     

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75-110 of influenza virus haemagglutinin) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding.     

Chemical synthesis of a polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product

We previously determined the nucleotide sequence of the 3' end of Moloney leukaemia virus and discovered the potential coding region for an unknown protein, R. We now show that this region does encode a protein. A pentadecapeptide of R was chemically synthesized and antibodies raised against it. Antisera to the synthetic peptide recognize the R protein and the env precursor polyprotein in infected cells. The strategy presented here should provide a general method for accessing proteins predicted by nucleotide sequences.     

Glycyl glutamine, an inhibitory neuropeptide derived from beta-endorphin

The primary mechanism of activation of intracellular prohormones seems to involve proteolytic cleavage at sequences of consecutive basic residues. Thus, all the known biologically active peptides derived from the prohormone of corticotropin and beta-endorphin appear to be excised initially by enzymes with this specificity. The C-terminal peptide, beta-endorphin (1-31), is generated by cleavage at a lysyl arginine sequence and an additional cleavage can give rise to the related peptides, beta-endorphin (1-27) and beta-endorphin (1-26). These derivatives of beta-endorphin are released by an endopeptidase that appears to catalyse cleavage on the carboxyl side of paired lysine residues, followed by the action of a carboxypeptidase B-like enzyme (Fig. 1). The beta-endorphin fragments, beta-endorphin (1-27) and beta-endorphin (1-26), have been isolated from porcine and bovine pituitary but the C-terminal dipeptide, glycyl glutamine, has not been reported previously. Here we describe the isolation of glycyl glutamine from porcine pituitary and present evidence for its presence in sheep brain stem. When applied ionophoretically to brain stem neurones in the rat, the dipeptide exhibited an inhibitory action on cell firing.     

Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein. Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein, and the epitopes recognized can be defined with short synthetic peptides in vitro. Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the H1 subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane.