Asparagine repeats are rare in mammalian proteins.
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Animals↗
Biomedical subjects
G Kreil
Publications and source records attributed to G Kreil.
The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes.
Bv8, a protein from skin secretions of Bombina variegata, reacts with receptors present in mammalian brain and intestine (Mollay et al. (1999) Eur. J. Pharmacol. 374, 189-196). As deduced from cloned cDNAs, the murine and human Bv8 homologues have identical amino-terminal sequences and also contain 10 cysteines. From mouse testes, two forms of Bv8 mRNA have been characterized, of which one contains an additional exon which codes for 21 mostly basic amino acids. The mouse Bv8 gene is most active in mid-late pachytene spermatocytes. In mouse testes, Bv8 mRNA can first be detected at the end of the second week post partum.
Amino Acid Sequence↗
Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats.
From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.
Amino Acid Sequence↗
Protirelin (thyrotropin-releasing hormone) in thyroid gland: possible involvement in regulation of thyroid status.
AIM: To establish the presence of the hypothalamic hormone protirelin (thyrotropin-releasing hormone, TRH) in human thyroid and to investigate whether the concentration of this peptide in the thyroid gland is sensitive to thyroid status. METHODS: A procedure has been developed for the determination of TRH in the thyroid gland, distinct from TRH-like peptides which also react with TRH-antibody. RESULTS: Human thyroid was shown to contain both authentic TRH and TRH-like peptides, a similar pattern was seen in a range of animal thyroids. The concentrations of TRH in non-active goiter thyroids were substantial (41.6-248 pmol.g-1); in contrast the thyroids from hyperthyroid patients contained very little TRH (0.01-2.52 pmol.g-1). CONCLUSION: The physiologic role of TRH in the thyroid is not known but the large difference between the concentrations of this hormone in non-active and hyperactive thyroids suggests that thyroidal TRH may be involved in the regulation of thyroid status.
Goiter↗
Molecular cloning in the marmoset shows that semenogelin is not the precursor of the TRH-like peptide pGlu-Glu-Pro amide.
Two peptides with similar structures to thyrotropin-releasing hormone (TRH), pGlu-Glu-Pro amide and pGlu-Phe-Pro amide, have been identified in human seminal fluid and it has been shown that one of these peptides, pGlu-Glu-Pro amide, has the ability to increase the capacitation of sperm cells, consistent with a role in fertility. In order to select a species in which there is a high degree of expression of the genes that code for 'TRH-like' peptides, we have determined the levels of these peptides in the prostate, pancreas and thyroid of a range of species including rat, rabbit, ox, marmoset, macaque and man. The peptides were extracted from the tissues and purified before determination by RIA with TRH antibody. In addition, trypsin digestion and TRH RIA was used to investigate the presence of N-extended forms. The highest concentrations of TRH-immunoreactive peptides were found in the tissues of the marmoset, Callithrix jacchus. Ion-exchange chromatography demonstrated that marmoset thyroid contained principally authentic TRH, the pancreas contained both TRH and TRH-like peptides while the prostate contained TRH-like peptides alone. Further purification by HPLC showed that the main TRH-immunoreactive peptide in marmoset prostate was pGlu-Glu-Pro amide and a second component was identified as pGlu-Phe-Pro amide. The results indicate that the biosynthesis of these peptides could be studied to advantage in the marmoset. The biosynthetic precursors of the TRH-like peptides have not been identified. To examine whether pGlu-Glu-Pro amide might originate from semenogelin, we determined the sequence of semenogelin in the marmoset. It exhibited a high degree of homology with human semenogelin-I, but in place of the Lys-Gln-Glu-Pro sequence that might give rise to pGlu-Glu-Pro amide, marmoset semenogelin possessed the sequence Ser-Gln-Asp-Gln which cannot serve as a precursor for a TRH-like peptide. Further evidence was obtained by Northern blot analysis of a range of marmoset tissues. The results showed that semenogelin is not present in marmoset prostate. It is concluded that pGlu-Glu-Pro amide originates from a precursor distinct from semenogelin, both in marmoset and in man.
Amino Acid Sequence↗
HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity.
Using Expressed Sequence Tags (ESTs) deposited in the data banks, a cDNA has been assembled that encodes a protein related to the hyaluronidases from bee venom and mammalian sperm. Expression of this cDNA yielded a polypeptide termed HYAL2, which is located in lysosomes. The HYAL2 protein was shown to have hyaluronidase activity below pH 4. However, it only hydrolyzed hyaluronan of high molecular mass from umbilical cord, rooster comb, and a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which was further hydrolyzed to small oligosaccharides by the sperm hyaluronidase. Conversely, hyaluronan fragments from vitreous humor, which had a molecular mass of about 20 kDa, were not cleaved by the HYAL2 enzyme to any detectable extent. These results provide evidence for the existence of structural domains in hyaluronan, which are resistant to the action of this enzyme. The structural and functional implications of these findings are discussed.
Amino Acid Sequence↗
A new opioid peptide predicted from cloned cDNAs from skin of Pachymedusa dacnicolor and Agalychnis annae.
We have isolated a cDNA encoding a precursor of dermorphin from the skin of Pachymedusa dacnicolor. Besides four copies of this opioid peptide, the deduced sequence also contains the genetic information for a novel peptide Tyr-Ile-Phe-His-Leu-Met-Asp-NH2. This differs from Met-deltorphin by the presence of Ile at position 2. In a related precursor from the skin of Agalychnis annae, the sequence of this peptide is in the 3'-untranslated region of the cloned cDNA. From earlier results we predict that in skin peptides the second residue is D-allo-Ile. We have synthesized this and related peptides with different D-amino acids, and determined their delta agonist activity. The peptide with D-nor-Leu binds with high affinity to delta receptors, while that with D-allo-Ile is about 100 times less active.
Amino Acid Sequence↗
The soluble hyaluronidase from bull testes is a fragment of the membrane-bound PH-20 enzyme.
The membrane-bound PH-20 hyaluronidase is known to be essential for fertilization. Here we addressed the question whether the soluble hyaluronidase from bull teste is related to the PH-20 polypeptide. The sequence of the membrane-bound PH-20 hyaluronidase from bovine sperm was determined via cDNA cloning. In parallel, from a commercial preparation of bovine hyaluronidase the major 60-kDa form was purified to apparent homogeneity. The soluble enzyme was digested with two different proteases and with cyanogen bromide and the amino acid sequence of 44 different fragments was determined. All the peptide sequences could be aligned to the sequence deduced from the cloned cDNAs. Our results thus show that the soluble 60-kDa hyaluronidase from bovine testes is a glycoprotein derived from the sperm PH-20 enzyme. As compared to the primary translation product of the PH-20 mRNA, it lacks the signal peptide at the amino terminus and 56 amino acids at the carboxyl end. These results demonstrate that the soluble 60-kDa enzyme is a fragment of the PH-20 hyaluronidase. It is currently not known whether the soluble testes hyaluronidase has a distinct biological function.
Amino Acid Sequence↗
In vitro mutagenesis of PH-20 hyaluronidase from human sperm.
The cDNA encoding PH-20 hyaluronidase from human sperm has been mutated at five positions by in vitro mutagenesis. We have changed three acidic amino acids and two arginine residues that are conserved in the sequence of mammalian PH-20 polypeptides as well as in the hyaluronidases from bee and hornet venom. Of the former, the mutants [Gln113]PH-20 and [Gln249]PH-20 had no detectable enzymatic activity; the mutant [Asn111]PH-20 had about 3% activity. The mutant [Thr252]PH-20 was also inactive, while [Gly176]PH-20 had only about 1% activity. This indicates that the PH-20 hyaluronidases, like numerous enzymes that hydrolyze glycosidic bonds, have acidic amino acids in their active site. Moreover, for the binding of the substrate, the polyanion hyaluronan, arginine residues appear to be essential.
Amino Acid Sequence↗
cDNA cloning and expression of secreted Xenopus laevis dipeptidyl aminopeptidase IV.
From a Xenopus laevis skin library a cDNA coding for dipeptidyl aminopeptidase IV (DPP IV) was isolated. The ORF codes for a protein with sequence similarity to DPP-IV-like proteins, including mammalian DPP IV and X. laevis fibroblast activation factor. In contrast to the membrane-bound mammalian enzymes, mature X. laevis DPP IV is a soluble secreted polypeptide. The frog enzyme possesses a cleavable signal sequence; the mature protein starts at Thr30 of the polypeptide predicted from the cDNA sequence. Expression of the cloned cDNA by recombinant vaccinia virus resulted in the formation of a protein with the expected molecular mass and substrate specificity. Recombinant DPP IV was present in high concentration in the supernatant of infected cells and exhibited enzymatic activity towards the synthetic substrate alanyl-prolyl-p-nitroanilide.
Amino Acid Sequence↗
Tachykinins and other biologically active peptides from the skin of the Costa Rican phyllomedusid frog Agalychnis callidryas.
Peptides present in a methanol extract prepared from skin of the Costa Rican frog Agalychnis callidryas of the Phyllomedusinae subfamily were studied by sequence analysis and pharmacological tests. Members of five different peptide families-tachykinins, bradykinins, caerulein, opioid peptides and sauvagine-were found. In particular, the extract contained a number of tachykinins with the following sequences: Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asp-Arg(Lys)-Phe-Tyr-Pro-Gly-Met-NH2, pGlu-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Pro-Gly-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Tyr-Pro-Val-Met. The latter three peptides have the unusual C-terminal sequence Pro-Gly(or Val)-Met-NH2 rather than Gly-Leu-Met-NH2 found in many other members of the tachykinin family. The observed amino acid substitutions may be the reason for the marked decrease in the biological activity observed in all in vitro and in vivo tests, even through the spectrum of tachykinin activities was retained. A kassinin-like peptide, with the sequence Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, was also found in the A. callidryas skin. While kassinin has a much higher affinity for NK-3 than for NK-1 receptors, the opposite is true for this A. callidryas peptide. The extract from A. callidryas skin also contained a new caerulein (pGlu-Asp-Tyr(HSO3)-Lys-Gly-Trp-Met-Asp-Phe-NH2) and a phyllokinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Tyr), as well as the opioid peptides dermorphin and [Hyp6]dermorphin, both previously isolated from different Phyllomedusa species.
Animals↗
D-amino acids in animal peptides.
D-amino acids have been detected in a variety of peptides synthesized by animal cells. These include opiate and antimicrobial peptides from amphibian skin, neuropeptides from snail ganglia, a hormone from crustaceans, and a constituent of a spider venom. cDNA cloning has shown that at those positions where a D-amino acid is found in the end-product, a normal codon for the corresponding L-amino acid is present. This implies that the D-residues are formed from L-amino acids by a posttranslational reaction. A prototype enzyme catalyzing such a reaction has recently been isolated from the venom of the funnel web spider.
Amino Acids↗
Hyaluronate-independent metastatic behavior of CD44 variant-expressing pancreatic carcinoma cells.
Several studies have demonstrated a correlation between the expression of CD44 variant isoforms and the ability of tumor cells to metastasize. The CD44 proteins carry amino acid sequence motifs that confer the ability to bind to the extracellular matrix component hyaluronate (HA). In this study, we investigated whether a CD44 variant previously shown to stimulate metastasis in a rat pancreatic carcinoma model (BSp73AS) is capable of binding to HA, and whether such binding is critical for metastasis. We show that transfection of this CD44 variant into BSp73AS cells increases the HA-binding capacity of the cells in a dose-dependent manner. Transfection of the same CD44 variant isoform into BDX2 cells also conferred strong HA-binding properties on these cells, but was insufficient to cause them to metastasize. Transfection of a surface-bound hyaluronidase into metastasizing BSp73AS cells bearing variant CD44 efficiently ablated the ability of these cells to bind to HA. However, in metastasis assays, these hyaluronidase-transfected cells showed patterns of metastasis similar to those of the parental cell line. We also show that the HA-binding capacity of a variety of tumor cells is not correlated with their metastatic proclivity, and that an antibody previously shown to block metastasis of the pancreatic carcinoma cells does not interfere with their ability to bind to HA. We conclude that although CD44 variant expression does promote metastasis formation, HA binding by tumor cells is not rate limiting for metastasis in the BSp73AS system and probably also in other metastasizing tumors. Furthermore, for metastasis by CD44 variant-expressing BSp73AS cells to occur, contact of the CD44 variant protein with a ligand other than HA Is required.
Amino Acid Sequence↗
Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan.
DG42 is one of the main mRNAs expressed during gastrulation in embryos of Xenopus laevis. Here we demonstrate that cells expressing this mRNA synthesize hyaluronan. The cloned DG42 cDNA was expressed in rabbit kidney (RK13) and human osteosarcoma (tk-) cells using a vaccinia virus system. Lysates prepared from infected cells were incubated in the presence of UDP-N-acetylglucosamine and UDP-[14C]glucuronic acid. This yielded a glycosaminoglycan with a molecular mass of about 200,000 Da. Formation of this product was only observed in the presence of both substrates. The glycosaminoglycan could be digested with testicular hyaluronidase and with Streptomyces hyaluronate lyase but not with Serratia chitinase. Hyaluronan synthase activity could also be detected in homogenates of early Xenopus embryos, and the activity was found to correlate with the expression of DG42 mRNA at different stages of development. Synthesis of hyaluronan is thus an early event after midblastula transition, indicating its importance for the ensuing cell movements in the developing embryo. Our results are at variance with a recent report (Semino, C. E. & Robbins, P. W. (1995) Proc. Natl. Acad. Sci. USA 92, 3498-3501) that DG42 codes for an enzyme that catalyzes the synthesis of chitin-like oligosaccharides.
Animals↗
The TRH-like peptides in rabbit testis are different from the TRH-like peptide in the prostate.
Human seminal fluid contains a number of tripeptide amides with similar structures to thyrotropin releasing hormone (TRH), two of which have been identified as pGlu-Glu-Pro amide and pGlu-Phe-Pro amide. To determine whether these peptides originate in the same tissues and have the same molecular origin, TRH-immunoreactive peptides were extracted from the prostate and testis of the rabbit, purified by ion exchange chromatography and HPLC, and identified by co-chromatography with 3H-labelled marker peptides. In addition, trypsin digestion was used to release TRH-like tripeptides from N-extended forms of these peptides. The sole TRH-like peptide in the prostate was shown to be pGlu-Glu-Pro amide; it was not accompanied by a detectable amount of pGlu-Phe-Pro amide. The prostate also appeared to contain a very small amount of N-extended forms of these peptides. In contrast to the prostate, the testis contained high concentrations of N-extended forms of pGlu-Phe-Pro amide but essentially no tripeptide. The testis also contained N-extended forms of two other neutral TRH-like peptides which were less hydrophobic than pGlu-Phe-Pro amide. Neither the prostate nor the testis contained a significant amount of TRH. The results show that in the rabbit the TRH-like peptides pGlu-Glu-Pro amide and pGlu-Phe-Pro amide occur in different tissues and appear to be formed from different precursors.
Amino Acid Sequence↗
BSTI, a trypsin inhibitor from skin secretions of Bombina bombina related to protease inhibitors of nematodes.
From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with K(i) values in the range of 0.1 to 1 microM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.
Amino Acid Sequence↗
The precursors of the bee venom constituents apamin and MCD peptide are encoded by two genes in tandem which share the same 3'-exon.
From a cDNA library prepared from venom glands of worker bees, clones encoding the precursors of apamin and MCD peptide have been isolated. The cDNAs are similar at the 5'-ends and identical in their 3'-regions. Analysis of the corresponding genes has revealed the existence of six exons separated by introns rich in A + T. Starting from the 5'-end, these exons are arranged in the following order: three exons of the mast cell-degranulating (MCD) peptide precursor, two exons of the gene for the apamin precursor, and finally a 3'-exon present in both cDNAs. This suggests that the bulk of the apamin gene resides in the third intron of the MCD peptide gene. Using inverse polymerase chain reaction, a segment of genomic DNA upstream of the first exon of the MCD precursor gene was obtained. The sequence of this segment shows 81% identity to the DNA sequence preceding the first exon of the apamin gene and both contain a putative TATA box. We thus propose that the mRNA encoding the apamin precursor originates from a primary transcript which starts in the third intron of the MCD peptide gene. Both cDNAs encode unusually small precursors comprising only 46 amino acids in case of apamin and 50 in the case of the MCD peptide.
Amino Acid Sequence↗