Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

[1]  P. Laming,et al.  Relationship between ecg, eeg and sps responses during arousal in the goldfish (Carassius auratus) , 1990 .

[2]  J. Aletta,et al.  Protein methylation: a signal event in post-translational modification. , 1998, Trends in biochemical sciences.

[3]  J. Veenstra.  Isolation and structure of two gastrin/CCK-like neuropeptides from the American cockroach homologous to the leucosulfakinins , 1989, Neuropeptides.

[4]  G. Gäde.  The explosion of structural information on insect neuropeptides. , 1997, Fortschritte der Chemie organischer Naturstoffe = Progress in the chemistry of organic natural products. Progres dans la chimie des substances organiques naturelles.

[5]  R. Kellner,et al.  Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana , 1997, Regulatory Peptides.

[6]  W. Haddon,et al.  Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin and cholecystokinin. , 1986, Science.

[7]  W. Huttner,et al.  Tyrosine sulfation and the secretory pathway. , 1988, Annual review of physiology.

[8]  J. Gordon,et al.  Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurrence. , 1986, Biochemical and biophysical research communications.

[9]  G. Gade,et al.  The explosion of structural information on insect neuropeptides. , 1997 .

[10]  D. Price,et al.  Relationships between the FMRFamide-related peptides and other peptide families , 1988, Peptides.

[11]  P. Bräunig,et al.  Comparative aspects of peptidergic signaling pathways in the nervous systems of arthropods. , 1995, EXS.

[12]  Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry , 1999, Regulatory Peptides.

[13]  W. Giard,et al.  Insect Myosuppressins and Sulfakinins Stimulate Release of the Digestive Enzyme α‐Amylase in Two Invertebrates: The Scallop Pecten maximus and Insect Rhynchophorus ferrugineus , 1997 .

[14]  J. Chung,et al.  Does the N-terminal pyroglutamate residue have any physiological significance for crab hyperglycemic neuropeptides? , 1996, European journal of biochemistry.

[15]  L. Hood,et al.  A gas-liquid solid phase peptide and protein sequenator. , 1981, The Journal of biological chemistry.

[16]  P. Proost,et al.  Isolation and primary structure of two sulfakinin-like peptides from the fleshfly, Neobellieria bullata. , 1992, Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology.

[17]  R. Nachman,et al.  Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly, Musca domestica , 1999, Peptides.

[18]  R. Kellner,et al.  Isolation and Structural Elucidation of Two Pyrokinins From the Retrocerebral Complex of the American Cockroach , 1997, Peptides.

[19]  P. Cooper,et al.  Distribution of sulfakinin-like peptides in the central and sympathetic nervous system of the American cockroach, Periplaneta americana (L.) and the field cricket, Teleogryllus commodus (Walker). , 1997, Tissue & cell.

[20]  W. Haddon,et al.  Structural aspects of gastrin/CCK-like insect leucosulfakinins and FMRF-amide , 1988, Peptides.

[21]  Á. Hernanz,et al.  Inhibition of Human Neutrophil Functions by Sulfated and Nonsulfated Cholecystokinin Octapeptides , 1997, Peptides.

[22]  J. Dixon,et al.  Identification and characterization of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin. , 1988, The Journal of biological chemistry.

[23]  W. Huttner,et al.  (Glu62, Ala30, Tyr8)n serves as high-affinity substrate for tyrosylprotein sulfotransferase: a Golgi enzyme. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. K. Hayes,et al.  Isolation and Identification of a Sulfakinin-Like Peptide, with Sequence Homology to Vertebrate Gastrin and Cholecystokinin, from the Brain of Locusta Migratoria , 1990 .

[25]  W. Haddon,et al.  Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin. , 1986, Biochemical and biophysical research communications.

[26]  J. Veenstra.  Isolation and Structure of Three Neuropeptides from the Corpora Cardiaca of the American Cockroach , 1990 .

[27]  J. Rehfeld,et al.  The Sulfakinins of the Blowfly Calliphora Vomitoria , 1995 .

[28]  W. Fischer,et al.  Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[29]  R. Kellner,et al.  Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach. , 1999, Insect biochemistry and molecular biology.

[30]  D. Fry,et al.  Analogs of CCK incorporating conformationally constrained replacements for Asp32. , 1992, Journal of medicinal chemistry.

[31]  R. Nichols,et al.  Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I , 1992, Molecular and Cellular Neuroscience.

[32]  L. Schoofs,et al.  Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources. , 1990, Comparative biochemistry and physiology. B, Comparative biochemistry.

[33]  J. Rehfeld,et al.  The sulfakinins of the blowfly Calliphora vomitoria. Peptide isolation, gene cloning and expression studies. , 1995, European journal of biochemistry.

[34]  J. Stock,et al.  A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. , 1996, Proceedings of the National Academy of Sciences of the United States of America.