Yukichi Abe

Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor.

The ubiquitin–proteasome system is essential for intracellular protein degradation, but an extracellular role of this system has not been known until now. We have previously reported that the proteasome is secreted into the surrounding seawater from sperm of the ascidian (Urochordata) Halocynthia roretzi on sperm activation, and that the sperm proteasome plays a key role in fertilization. Here, we show that a 70-kDa component (HrVC70) of the vitelline coat is the physiological substrate for the ubiquitin–proteasome system during fertilization of H. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) was isolated, and a homology search revealed that HrVC120 contains 13 epidermal growth factor-like repeats and a mammalian zona pellucida glycoprotein-homologous domain. HrVC70 functions as a sperm receptor. We demonstrate that HrVC70 is ubiquitinated both in vitro and in vivo. The immunocytochemical localization of multiubiquitin chains in the vitelline coat and the inhibitory effect of monoclonal antibodies against the multiubiquitin chains on fertilization strongly support the role of the ubiquitin–proteasome system in ascidian fertilization. Taken together, these results indicate that the ubiquitin–proteasome system is responsible for extracellular degradation of the sperm receptor HrVC70 and, consequently, for sperm penetration of the vitelline coat during fertilization.

Multiple forms in the subunit structure of concanavalin A

Abstract Concanavalin A, a phytohemagglutinin from jack beans, has at least two forms in the subunit structure. One of them is made up by a single subunit species of molecular weight 27·103 (probably a dimer at pH 5). The other may consist of two kinds of subunits (tetramer), both of which have molecular weight of about a half of the above-mentioned value. The presence of the third structure containing all the three subunits cannot be excluded. Chemical studies on the isolated subunit proteins suggest that the two smaller subunits have a close structural relation to the two fragments, respectively, arising from a polypeptide of the larger subunit when divided into nearly equal sizes.

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi: Isolation and characterization

Abstract The lectin from the hemolymph of solitary ascidian, Halocynthia roretzi, has been isolated in an electrophoretically homogeneous form by affinity chromatography on a column of acid-treated Sepharose. It is a large protein with s20,w=24 S, composed of subunits with a molecular weight of 41,000. D-Galactose and various disaccharides containing D-galactose inhibit the hemagglutinating activity of the lectin. Thus, H. roretzi lectin is a D-galactose-specific lectin.

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi. Molecular, binding and functional properties

Galactose-specific lectin isolated from the hemolymph of solitary ascidian, Halocynthia roretzi, has been further characterized. The hemagglutinating activity of the lectin is Ca2+-dependent. The lectin has a large molecular form as revealed by gel-permeation chromatography, sedimentation equilibrium and velocity measurement, and electron microscopic observation. The lectin is adsorbed to columns of blue-Sepharose and phenyl-Sepharose, and eluted with ethylene glycol, not with lactose or high concentration of NaCl. The lectin shows a stimulatory effect on the superoxide anion production by guinea-pig polymorphonuclear leukocytes, and the effect is inhibited, among various sugars, most strongly by melibiose.

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.

EFFECTS OF SUCCINYLATED CONCANAVALIN A ON INFECTIVITY AND SYNCYTIAL FORMATION OF HUMAN IMMUNODEFICIENCY VIRUS

The effects of various lectins on the infectivity of human immunodeficiency virus (HIV) type 1 was investigated. Among the 25 lectins investigated, 2 types of concanavalin A (Con A) and 3 types of phytohemagglutinin were found to inhibit HIV infection. Succinylated Con A (S-Con A) efficiently blocked HIV-induced formation of syncytia in a coculture of MOLT-4 cells and blocked cell-free infection by HIV of MT-4 cells. The HIV-binding study revealed that S-Con A only partially inhibited viral binding to cells, although the control Leu-3a monoclonal antibody strongly inhibited it. When S-Con A was added to cultures after the initiation of viral adsorption, the number of HIV antigen-positive cells that developed depended on the time interval before addition of the compound. S-Con A inhibited HIV infection even after viral binding to cells at 0 °C and further incubation at 37 °C for 1 day. These data suggest that S-Con A inhibited mainly the fusion process rather than viral binding to cells in exerting its anti-HIV activity.

Primary structure and function of superoxide dismutase from the ascidian Halocynthia roretzi

A protein with a molecular weight of 17K, immunoreactive with the S-1B2 antibody, has been isolated from hemocytes of Halocynthia roretzi. Its amino acid sequence has been determined by sequential Edman degradation analysis of peptide fragments derived from proteolytic fragmentation. The 17K protein is a single chain protein consisting of 151 amino acids with an acylated N-terminal serine. A comparison of the amino acid sequence of H. roretzi 17K protein with those of other proteins reveals that the 17K protein is Cu,Zn-SOD. The protein was found to have a KCN-inhibited SOD activity. Cu,Zn-SOD has been purified from H. roretzi plasma. The molecular weight is 17K and the activity is inhibited with KCN and diethyldithiocarbamate. It has been demonstrated that it can enhance phagocytosis by H. roretzi hemocytes. Thus, plasma Cu,Zn-SOD plays a role in H. roretzi as a defense molecule.

Alteration of quaternary structure and biological activity of concanavalin A

A major molecular species of concanavalin A (Con A), a mitogenic lectin from jack bean seeds, has a quaternary structure composed of four homologous subunits and a tetravalent sugar-binding ability. We show that the tetrameric Con A can be converted into a monovalent monomeric form by either photochemical alkylation or hydrogen peroxide/dioxane oxidation of about two tryptophan residues. A divalent dimeric derivative of Con A is also prepared by sulfomethylamidation of about four carboxyl groups. Chemical properties and mitogenic and hemagglutinating activities of these new Con A derivates are compared with those of the tetravalent Con A, as well as of the Con A derivatives that have appeared in the literature on cell biological studies. The significance of the lectin valences in lymphocyte activation and hemagglutination is also discussed.

High-performance affinity chromatograhy of concanavalin A

Abstract d -Glucose was immobilized as a form of maltamyl group on Asahipak GS-520, a synthetic polymer-type gel, and employed as an affinity adsorbent for high-performance affinity chromatography. The method proved useful for the rapid fractionation of tetravalent and divalent molecular species contained in the usual preparations of concanavalin A. It also afforded an adequate means for the efficient purification of a monovalent derivative of concanavalin A produced by a photochemical reaction.

A Novel Protease from Jack-Bean Seeds: Asparaginyl Endopeptidase

Enzymatical and chemical reactions that induce the peptide-bond cleavage at definite amino acid residues and so produce a limited number of fragments from proteins are essential tools in protein sequence analysis. Although several kinds of residue-specific proteases are now widely used for this purpose, the asparaginyl-bond specific protease has not been available yet. Carrington et al. (1985) and Bowles et al. (1986) have reported that concanavalin A, a lectin of jack bean (Canavalia ensiformis), is produced by post-translational proteolysis and transpeptidation (or ligation) at the carboxyl side of Asn-residues of its precursor. Their results suggest that the bean at least in its premature state contains an asparaginyl endopeptidase which may have a dual function, proteolysis and transpeptidation. We confirmed this suggestion by isolating it from commercially available jack-bean meal and showed its high utility in protein sequence analysis. Purification and characterization of this novel protease are described in the following.