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.

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.

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus PhyUomedusa, that have a higher affinity and selectivity for 6 opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of PhyUomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, deltorphins I and II, show an even higher affinity for 6 receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phylomedusa skin, dermorphin, which is highly selective for ,u-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both pA and 6 opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.The endogenous opioid ligands isolated from vertebrate brain show little selectivity toward the different types of opioid receptors. Peptides isolated from amphibian skin appear to be more selective. In 1981 Montecucchi et al. (1) extracted from the skin of the Argentinian frog Phyllomedusa sauvagei a heptapeptide named dermorphin, which preferentially binds to A-type opioid receptors (2). By recombinant DNA techniques, it was demonstrated that dermorphin, like numerous other peptides, is derived in multiple copies from larger precursors. In addition, from inspection of the sequence of one of the cloned cDNAs for these precursors, the existence of another heptapeptide with an N-terminal region similar to that of dermorphin was predicted (3). We recently succeeded in isolating small quantities of this peptide from the skin ofP. sauvagei and named it deltorphin, because of its high affinity and selectivity for the 8 opioid binding site (4). Both dermorphin and deltorphin contain a D amino acid (D-alanine and D-methionine, respectively) as the second amino acid. In the cloned cDNAs, codons for the corresponding L amino acids-i.e., GCG for alanine and ATG for methionine-were found at these positions. This characteristic suggested that the processing of these peptides includes a reaction whereby an L amino acid residue is converted to its D isomer within peptide linkage (3). Here we describe the isolation of two other heptapeptides from the skin of Phyllomedusa bicolor, which show an affinity and selectivity for 8 opioid receptors several times higher than that of deltorphin and the cyclic enkephalin derivative enkephalin (DPDPE, where D-Pen is D-penicillamine) (5). Once again, these peptides contain a D-alanine residue in the second position and share with dermorphin and deltorphin the N-terminal sequence Tyr-D-Xaa-Phe. We refer to these peptides, which differ by the presence of an aspartic or glutamic residue in position 4,...

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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.