Hans Jörnvall

Galanin - a novel biologically active peptide from porcine intestine

The isolation of a novel biologically active peptide, designated galanin, is described. The peptide was discovered by the detection of its C‐terminal amide structure in porcine intestinal extract using a chemical method. It was found that galanin consists of 29 amino acids and the complete amino acid sequence is: contract smooth muscle preparations from the rat and to cause a mild and sustained hyperglycemia in dog.

Short-chain dehydrogenases/reductases (SDR).

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family. Presently, at least 57 characterized, highly different enzymes belong to this family and typically exhibit residue identities only at the 15-30% level, indicating early duplicatory origins and extensive divergence. In addition, another family of 22 enzymes with extended protein chains exhibits part-chain SDR relationships and represents enzymes of no less than three EC classes. Furthermore, subforms and species variants are known of both families. In the combined SDR superfamily, only one residue is strictly conserved and ascribed a crucial enzymatic function (Tyr 151 in the numbering system of human NAD(+)-linked prostaglandin dehydrogenase). Such a function for this Tyr residue in SDR enzymes in general is supported also by chemical modifications, site-directed mutagenesis, and an active site position in those tertiary structures that have been characterized. A lysine residue four residues downstream is also largely conserved. A model for catalysis is available on the basis of these two residues. Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecules, where a common GlyXXXGlyXGly pattern occurs. Two SDR enzymes established by X-ray crystallography show a one-domain subunit with seven to eight beta-strands. Conformational patterns are highly similar, except for variations in the C-terminal parts. Additional structures occur in the family with extended chains. Some of the SDR molecules are known under more than one name, and one of the enzymes has been shown to be susceptible to native, chemical modification, producing reduced Schiff base adducts with pyruvate and other metabolic keto derivatives. Most SDR enzymes are dimers and tetramers. In those analyzed, the area of major subunit contacts involves two long alpha-helices (alpha E, alpha F) in similar and apparently strong subunit interactions. Future possibilities include verification of the proposed reaction mechanism and tracing of additional relationships, perhaps also with other protein families. Short-chain dehydrogenases illustrate the value of comparisons and diversified research in generating unexpected discoveries.

Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue

Abstract A heptacosapeptide with potent gastrin releasing activity has been isolated from porcine non-antral gastric and intestinal tissue. The amino acid sequence suggested from a preliminary study on the gastric peptide is: Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2. Striking homology in the C-terminal region is seen with bombesin, accounting for the similar bioactivities of the two peptides. Some structural resemblance with porcine cholecystokinin in the N-terminal region is noted.

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo‐epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo‐epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30‐positive cells appear in the ‘apoptotic’ subG1 peak. Tests with synthetic peptides define positions 387–396 of CK18, with a liberated C‐terminus at the caspase cleavage site DALD‐S, as the ten‐residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo‐epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin‐fixed material. Copyright

Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

Abstract.Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an α/β folding pattern with a central beta sheet flanked by 2–3 α-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

N-terminally extended somatostatin: The primary structure of somatostatin-28

Somatostatin, first isolated from ovine hypothalamic extracts [l], has been characterized as a tetradecapeptide [2], and the same peptide has been obtained from porcine hypothalamus [3]. Somatostatin-like immunoreactivity and bioactivity are widely distributed in the central nervous system and in the digestive tract tissues [4-71. Size heterogeneity described in somatostatin-like immunoreactive materials [3,6,8,9] raised the possibility of the existence of prohormonal forms of this molecule [lO,l 11. During the purification of porcine intestinal somatostatin differences were noticed in the chromatographic behaviour of immunoreactive fractions and synthetic somatogtatin-14 [ 121. A novel intestinal peptide was then isolated and partly characterized as an N-terminally extended form of somatostatin [ 131. We report here the primary structure of this intestinal peptide which has been found to be an octacosapeptide, somatostatin-28. The results establish size heterogeneity in the somatostatin family of peptides. Sequence comparison between somatostatin-28 and prohormones also suggest that it may be a precursor of somatostatin-14.

Neuropeptide K: Isolation, structure and biological activities of a novel brain tachykinin

A 36 amino acid residue peptide, which contains a substance K sequence at its C-terminus has been isolated from porcine brain extracts. The primary structure of the peptide, designated neuropeptide K (NPK), was found to be: (sequence; see text) This N-terminally extended form of substance K is present in a high concentration in the brain. The peptide is highly biologically active with regard to gallbladder contraction, protein extravasation, hypotension and bronchial smooth muscle spasm and may act as an additional tachykinin neuromessenger.

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK‐lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK‐lysin. The clone was used to show that stimulation with human interleukin‐2 induced synthesis of NK‐lysin‐specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin‐2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK‐lysin was present in cells positive for CD8, CD2 and CD4. NK‐lysin showed high anti‐bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK‐sensitive mouse tumour cell line, YAC‐1, but it did not lyse red blood cells. The amino acid sequence of NK‐lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK‐lysin is a new effector molecule of cytotoxic T and NK cells.

Short-chain dehydrogenase/reductase (SDR) relationships: A large family with eight clusters common to human, animal, and plant genomes

The progress in genome characterizations has opened new routes for studying enzyme families. The availability of the human genome enabled us to delineate the large family of short‐chain dehydrogenase/reductase (SDR) members. Although the human genome releases are not yet final, we have already found 63 members. We have also compared these SDR forms with those of three model organisms: Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana. We detect eight SDR ortholog clusters in a cross‐genome comparison. Four of these clusters represent extended SDR forms, a subgroup found in all life forms. The other four are classical SDRs with activities involved in cellular differentiation and signalling. We also find 18 SDR genes that are present only in the human genome of the four genomes studied, reflecting enzyme forms specific to mammals. Close to half of these gene products represent steroid dehydrogenases, emphasizing the regulatory importance of these enzymes.

The SDR (Short-Chain Dehydrogenase/Reductase and Related Enzymes) Nomenclature Initiative

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with presently over 46,000 members. In phylogenetic comparisons, members of this superfamily show early divergence where the majority have only low pairwise sequence identity, although sharing common structural properties. The SDR enzymes are present in virtually all genomes investigated, and in humans over 70 SDR genes have been identified. In humans, these enzymes are involved in the metabolism of a large variety of compounds, including steroid hormones, prostaglandins, retinoids, lipids and xenobiotics. It is now clear that SDRs represent one of the oldest protein families and contribute to essential functions and interactions of all forms of life. As this field continues to grow rapidly, a systematic nomenclature is essential for future annotation and reference purposes. A functional subdivision of the SDR superfamily into at least 200 SDR families based upon hidden Markov models forms a suitable foundation for such a nomenclature system, which we present in this paper using human SDRs as examples.