Günther Kreil

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.

Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for δ opioid receptors

With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for delta opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2.

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.

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.

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.

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.

The AVIT protein family

Homologues of a protein originally isolated from snake venom and frog skin secretions are present in many vertebrate species. They contain 80–90 amino acids, 10 of which are cysteines with identical spacing. Various names have been given to these proteins, such as mamba intestinal protein 1 (MIT1), Bv8 (Bombina variegata molecular mass ∼8 kDa), prokineticins and endocrine‐gland vascular endothelial growth factor (EG‐VEGF). Their amino‐terminal sequences are identical, and so we propose that the sequence of their first four residues, AVIT, is used as a name for this family. From a comparison of the sequences, two types of AVIT proteins can be discerned. These proteins seem to be distributed widely in mammalian tissues and are known to bind to G‐protein‐coupled receptors. Members of this family have been shown to stimulate contraction of the guinea pig ileum, to cause hyperalgesia after injection into rats and to be active as specific growth factors. Moreover, the messenger RNA level of one of these AVIT proteins changes rhythmically in the region of the brain known as the suprachiasmatic nucleus. This shows that members of this new family of small proteins are involved in diverse biological processes.

Enhancement of bee venom phospholipase A2 activity by melittin, direct lytic factor from cobra venom and polymyxin B☆

The peptide melittin and the enzyme phospholipase AZ, two main constituents of bee venom, show a marked synergism in their action on cell membranes. Under conditions where neither of the two substances has any deleterious effect, their combined action readily leads to the lysis of e.g. erythrocytes [l] . We have recently observed that mehttin forms a complex with lecithins provided the lipid is in the liquid-crystalline state [2j. This hydrophobic binding of melittin to phospholipids may be an intermediate step in the action of bee venom on cell membranes and thus be of relevance for explaining the synergistic action of venom components. If this argument is correct, one would expect that melittin stimulates the action of phospholipase only when complex formation between the peptide and the phospholipid can occur. In this report we present our results on the action of purified phospholipase AZ from bee venom on lecithin liposomes in the presence and absence of melittin. It could be shown that melittin stimulates the enzymatic hydrolysis of egg lecithin up to about 5-fold, while similar effects could not be observed with dipalmitoyllecithin. Previous experiments have shown that at the assay temperature melittin interacts with the former but not with the latter type of phospholipid. Starting from these findings, we have tested additional peptides known to interact with membranes: these

Nociceptive sensitization by the secretory protein Bv8

The small protein Bv8, isolated from amphibian skin, belongs to a novel family of secretory proteins (Bv8‐Prokineticin family, SWISS‐PROT: Q9PW66) whose orthologues have been conserved throughout evolution, from invertebrates to humans. When injected intravenously or subcutaneously (from 0.06 to 500 pmol kg−1) or intrathecally (from 6 fmol to 250 pmol) in rats, Bv8 produced an intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws. Topically delivered into one rat paw, 50 fmol of Bv8 decreased by 50% the nociceptive threshold to pressure in the injected paw without affecting the threshold in the contralateral paw. The two G‐protein coupled prokineticin receptors, PK‐R1 and PK‐R2, were expressed in rat dorsal root ganglia (DRG) and in dorsal quadrants of spinal cord (DSC) and bound Bv8 and the mammalian orthologue, EG‐VEGF, with high affinity. In DSC, PK‐R1 was more abundant than PK‐R2, whereas both receptors were equally expressed in DRG. IC50 of Bv8 and EG‐VEGF to inhibit [125I]‐Bv8 binding to rat DRG and DSC were 4.1±0.4 nM Bv8 and 76.4±7.6 nM EG‐VEGF, in DRG; 7.3±0.9 nM Bv8 and 330±41 nM EG‐VEGF, in DSC. In the small diameter neurons (<30 μm) of rat DRG cultures, Bv8 concentrations, ranging from 0.2 to 10 nM, raised [Ca2+]i in a dose‐dependent manner. These data suggest that Bv8, through binding to PK receptors of DSC and primary sensitive neurons, results in intense sensitization of peripheral nociceptors to thermal and mechanical stimuli.

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.