The human sperm protein PH-20 has hyaluronidase activity

AbstractThe 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

PII: S0968-0004(01)01835-7

Research Update We anticipate that further experimental investigation of the specific role of each of the five classes of Brix domain proteins in eukaryote model organisms, especially in yeast, will yield valuable new insights about ribosome synthesis and be key to our understanding of the ribosome biogenesis pathway. Acknowledgements We are grateful to E. Bogengruber, M. Breitenbach, F.M. Jantsch and G. Lepperdinger for supplying experimental data on sequence and function of Brix (AF319877) and yol077c before publication and for extensive discussion of the Brix sequence analysis results. This research was supported by Boehringer-Ingelheim International. Ubiquitin is a small protein, highly conserved among eukaryotes, that becomes covalently attached to both itself and a variety of cellular proteins 1,2 . The role of this ubiquitination is mostly to target proteins to the 26S proteasome degradation pathway 3 . In some cases, monoubiquitination (e.g. of histones) does not lead to degradation, but instead regulates other cellular processes such as chromatin remodeling 4 . Recently, several reports have described a role for monoubiquitination in a different pathway of protein degradation -the endocytosis and subsequent proteolysis of receptors and other transmembrane proteins by the vacuole or the lysosome 5,6 . According to the current model, the decisions about which protein is to be degraded at a specific time is made by the ubiquitination machinery, often in response to a prior event such as phosphorylation. Consequently, both the proteasome and the endocytosis machinery need a mechanism by which to faithfully recognize ubiquitinated proteins. The 26S proteasome comprises two main particles: the 20S core proteasome and the 19S regulatory complex. Subunit S5a (also known as Rpn10) of the 19S regulator binds polyubiquitin chains and has a preference for chains containing four or more ubiquitin monomers. The ubiquitin-interacting region has been mapped to two short, related motifs that are found in all members of the S5a family 7 . Using these regions, which comprise ~20 residues, as a starting point, we searched for other potential ubiquitin-binding sequences. Specifically, we used a combination of iterative database searches with generalized profiles, and Hidden Markov Models (profile-HMMs) 8 . Only sequences that matched a profile or an HMM derived from previously established family members, with error probabilities of p < 0.01, were used for subsequent iteration cycles. After eight cycles, the sequence motif 4 Migeon, J.C. et al. (1999) converged to a set of proteins shown in An observation of particular interest is the occurrence of UIMs in four classes of proteins involved in receptor endocytosisthe Eps15 subfamily of EH-domain proteins, the epsin subfamily of ENTH-domain proteins and two families of VHS-domain proteins, including the FYVE-finger proteins HRS and Vps27, and the SH3-domain proteins STAM and HBP. Eps15 is phosphorylated on Tyr850 by the ligandactivated epidermal growth factor (EGF) receptor and this phosphorylation is required for subsequent receptor endocytosis 10 . Furthermore, Eps15 binds to epsin, and both of these proteins interact with components of the endocytosis machinery, including clathrin and the AP-2 complex 11,12 . The fact that the phosphorylation site of Eps15 is immediately adjacent to a tandem UIM suggests that this motif might be involved in the regulated endocytosis of the EGF receptor. Liquid facets (lqf), an epsin from Drosophila melanogaster, was identified in a genetic screen as a dominant enhancer of the fat facets (faf) mutant eye phenotype 13 . This relationship links the gene encoding lqf with the ubiquitin system because faf is a deubiquitinating enzyme. An additional link between Eps15 and ubiquitin recognition is provided by the yeast protein Ede1p, the closest homolog of mammalian Eps15 (Ref. 14): in the yeast protein, the UIM is replaced by a UBA domain, a homology domain known to bind ubiquitin 15 . Proteins with an N-terminal VHS domain can be divided into three subtypes on the basis of their domain organization: (1) Vps27-HRS-like (type A); (2) STAM-HBP-like (type B); and (3) other proteins (type C) (se

Substrate specificity of a peptidyl-aminoacyl-l/d-isomerase from frog skin

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids

Bombinins, antimicrobial peptides from Bombina species

The skin secretions of Bombina species contain peptides and small proteins with interesting biological properties. These include bombesin, thyrotropin releasing hormone, BSTI and Bv8. In this review, the biosynthesis and antimicrobial activity of two groups of peptides, bombinins and bombinins H, are described. To date, these have only been found in Bombina skin. They are derived from common precursors containing one or two bombinin copies at the amino and a single bombinin H at the carboxyl end. Bombinins are active against Gram-positive and Gram-negative bacteria and fungi but virtually inactive in haemolysis assays. Conversely, bombinins H have lower bactericidal activities but lyse erythrocytes. In the skin secretions, bombinins H are present in two sizes with either 20 or 17 amino acids. Moreover, they occur as epimers with either an L- or a D-amino acid at position 2. An enzyme catalyzing this inversion of chirality of an amino acid in peptide linkage has been isolated from Bombina skin secretions. in different tests, also with different stages of the life cycle of Leishmania parasites, the D-forms were found to be more active. Biophysical studies have yielded some insight into the different behaviours of the epimers in model membranes. (C) 2009 Elsevier B.V. All rights reserved

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

AbstractA 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

Single amino acid repeats in signal peptides

There has been an increasing interest in single amino acid repeats ever since it was shown that these are the cause of a variety of diseases. Although a systematic study of single amino acid repeats is challenging, they have subsequently been implicated in a number of functional roles. In general surveys, leucine runs were among the most frequent. In the present study, we present a detailed investigation of repeats in signal peptides of secreted and type I membrane proteins in comparison with their mature parts. We focus on eukaryotic species because single amino acid repeats are generally rather rare in archaea and bacteria. Our analysis of over 100 species shows that repeats of leucine (but not of other hydrophobic amino acids) are over-represented in signal peptides. This trend is most pronounced in higher eukaryotes, particularly in mammals. In the human proteome, although less than one-fifth of all proteins have a signal peptide, approximately two-thirds of all leucine repeats are located in these transient regions. Signal peptides are cleaved early from the growing polypeptide chain and then degraded rapidly. This may explain why leucine repeats, which can be toxic, are tolerated at such high frequencies. The substantial fraction of proteins affected by the strong enrichment of repeats in these transient segments highlights the bias that they can introduce for systematic analyses of protein sequences. In contrast to a general lack of conservation of single amino acid repeats, leucine repeats were found to be more conserved than the remaining signal peptide regions, indicating that they may have an as yet unknown functional role