Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.

D-alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor.

A D-alanine-containing peptide termed dermorphin, with potent opiate-like activity, has been isolated from skin of the frog Phyllomedusa sauvagei. Complementary DNA (cDNA) libraries were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of an L-amino acid to its D-isomer.

Processing of precursors by dipeptidylaminopeptidases: a case of molecular ticketing.

Stepwise cleavage of dipeptides by dipeptidylaminopeptidases as a late step in the liberation of peptides from their precursors has been observed in diverse organisms such as yeast, insects and frogs.

The Brix domain protein family -- a key to the ribosomal biogenesis pathway?

Six (one archaean and five eukaryotic) protein families have similar domain architecture that includes a central globular Brix domain, and optional N- and obligatory C-terminal segments, both with charged low-complexity regions. Biological data for some proteins in this superfamily suggest a role in ribosome biogenesis and rRNA binding.

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.

Action of phospholipases on the cytoplasmic membrane of Escherichia coli. Stimulation by melittin.

The emission maximum of the single tryptophan residue of melittin was measured in the presence of phosphatidylethanolamine liposomes and Escherichia coli cytoplasmic membranes. In both cases, the fluorescence maximum was shifted to shorter wavelengths indicating a transfer of the indole ring to an apolar environment. E. coli membranes were labelled in position 2 of their phospholids with [14C]oleic acid. These membranes were used for measuring the activity of an endogenous phospholipase A2. A slow hydrolysis is observed, which can be accelerated by adding melittin. The extent of the stimulation depends on the molar ratio of melittin to membrane phospholipid. Under suitable conditions, the initial rate of hydrolysis is six to seven times higher in the presence than in the absence of melittin. The action of the phospholipase A2 from bee venom is also stimulated by melittin. An identical stimulation was observed with either E. coli membranes or pure phosphatidylethanolamine liposomes as substrate.

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.

Hyaluronidases--a group of neglected enzymes.

Hyaluronan is an important constituent of the extracellular matrix. This polysaccharide can be hydrolyzed by various hyaluronidases that are widely distributed in nature. The structure of some bacterial and animal enzymes of this type has recently been elucidated. It could be shown that the hyaluronidases from bee and hornet venom and the PH-20 hyaluronidase present on mammalian spermatozoa are homologous proteins.

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.

Hyal2--less active, but more versatile?

Hyal2 is one of several hyaluronidases present in vertebrates. The human gene encoding this enzyme is present on chromosome 3p.21.3, close to two additional hyaluronidase genes. cDNAs encoding Hyal2 homologues have been characterized from mouse and Xenopus laevis. These enzymes hydrolyze high molecular mass hyaluronan to intermediates of approximately 20 kDa, a finding which implies that structural domains of this size exist in this polysaccharide which was mostly thought to be a random coil. Hyal2 enzymes have an acidic pH-optimum with an activity that is considerably lower than observed for other types of hyaluronidases. Originally considered to be a typical lysosomal enzyme, more recent evidence has shown that Hyal2 proteins can also be exposed on the cell surface bound to the plasma membrane via a GPI anchor. Hyal2 is present in many tissues, one exception being the adult brain. In this tissue, the gene is silenced after birth by methylation. Current evidence about the role of Hyal2 in tumor growth, inflammation and frog embryogenesis is discussed.

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum.

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.

The constituents of storage granules in the dermal glands of Xenopus laevis. Structure of a basic polypeptide deduced from cloned cDNA.

Mature dermal glands of Xenopus laevis contain storage granules with a characteristic ellipsoid shape. A few major proteins are present in these granules, including two with the same amino-terminal sequence and apparent molecular masses of 26 and 28 kDa. Using antibodies against these proteins, positive clones were isolated from a cDNA expression library prepared from skin of X. laevis. One cDNA encodes a preprotein with a typical signal sequence and a mature part of 187 amino acids. The protein shows internal homology at both the amino and carboxyl end. The latter part has a very high content of basic amino acids.

Molecular cloning of a cDNA encoding the bombesin precursor in skin of Bombina variegata.

A cDNA library was constructed using poly(A)-rich RNA isolated from skin of the frog Bombina variegata. This library was screened with two oligo-nucleotides complementary to parts of the sequence of bombesin. The nucleotide sequence of one of the cloned cDNAs encoding a bombesin precursor is presented. The predicted polypeptide contains a single copy of the end-product. The bombesin sequence is preceded by a leucine residue suggesting an unusual type of precursor processing.

The human sperm protein PH-20 has hyaluronidase activity.

The PH-20 protein present on the membrane of guinea pig sperm was characterized using a monoclonal antibody [(1991) J. Cell Biol. 111, 2939-2949]. We have isolated the cDNA encoding the human PH-20 protein from a testis library. This cDNA was expressed in RK 13 cells using a vaccinia virus expression system. Cells expressing the human PH-20 protein possess hyaluronidase activity. Treatment with PI-PLC releases the hyaluronidase into the the medium with a concomitant large increase in enzymatic activity. These results demonstrate that the human PH-20 protein has hyaluronidase activity.

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.

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.

Dermal glands of Xenopus laevis contain a polypeptide with a highly repetitive amino acid sequence.

Mature dermal glands of Xenopus laevis contain storage granules with a characteristic ellipsoid shape. These granules contain, as a minor component, a heat-stable, acidic polypeptide with an apparent molecular mass of 75 kDa. Using antibodies against this protein, positive clones were isolated from a cDNA expression library prepared from skin of X. laevis. One of the cloned cDNAs encodes a pre-protein with a typical signal sequence and a mature part of 396 amino acids. The protein contains 33 copies of the sequence Gly-Gly/Glu-(Ala-Pro)2-4-Ala-Glu. Using the single-letter code for the four predominant amino acids, we have termed this polypeptide the APEG protein. Near its carboxy-terminus, one segment has been found with an amino acid sequence similar to that of spasmolytic polypeptide from porcine pancreas and to the human protein pS2.

A cDNA from brain of Xenopus laevis coding for a new precursor of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is found in large amounts in the skin of Xenopus laevis. In this tissue, 3 TRH precursor mRNAs can be detected of which the 2 more expressed encode almost identical proteins. However, Northern blot analysis of TRH precursor mRNAs in the brain of X. laevis revealed the existence of a new mRNA of about 1200 nucleotides which was present along with the larger TRH precursor mRNA identified in the skin. A cloned cDNA of a TRH precursor, corresponding in size to this new mRNA, was isolated and sequenced from a Xenopus brain lambda gt11 library. It encodes a precursor polypeptide which also contains 7 copies of TRH. However, at the amino acid level it differs by about 16% from the corresponding prepro-TRHs from skin. We have also attempted to characterize the gene encoding this prepro-TRH from Xenopus brain. Only the first and part of the second exon could be detected which are separated by an intron containing more than 8000 base pairs. Interestingly, the 5'-flanking region of this gene does not contain the characteristic promoter elements of the mammalian TRH genes suggesting marked differences in the regulation of their expression.

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.

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.