A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids

A novel glycosphingolipid-degrading enzyme was found in the cultured supernatant of Rhodococcus sp. G-74-2. It was purified 34.7-fold from the supernatant with 32.2% recovery by ammonium sulfate precipitation followed by Sephadex G-100 chromatography. The enzyme was demonstrated capable of cleaving the linkage between the oligosaccharide and ceramide of various acidic and neutral glycosphingolipids, producing intact oligosaccharides and ceramides. However, it was noted to hardly make any attack on linkages between monosaccharides and ceramides (cerebrosides) or between oligosaccharides and diacylglycerol (glycoglycerolipids). The enzyme preparation was completely free from various exoglycosidases and proteases. Furthermore, it was found to degrade neither N-linked nor O-linked glycoproteins. This enzyme, which is tentatively called endoglycoceramidase, should greatly facilitate the study of glycosphingolipids.     

A cave leech (Hirudinea, Erpobdellidae) from Croatia with unique morphological features

Croatobranchus mestrovi is a troglobitic leech from deep shaft-like caves in the Velebit Mountain, Dinaric karst, Croatia, living in cold (4–6 °C) water. Its oral sucker extends to form four pairs of triangular tentacles, each with about five finger-like papillae, but widening into a marginally crenulated disc when attached to a substrate. Pairs of stiff, finger-like lateral projections, probably gills, occur along the body behind the clitellum. Somites are simple five-annulate. Despite the unique head morphology and the presence of lateral outgrowths, the anatomy and 18S rRNA gene sequence of this species indicate that it is a member of the Erpobdellidae, closely related to Dina .     

Isolation and characterization of ceramide glycanase from the leech, Macrobdella decora

We have devised a simple method for achieving 890-fold purification of ceramide glycanase with 17% recovery from a North American leech, Macrobdella decora. The method includes water extraction, ammonium sulfate fractionation, and chromatography on octyl-Sepharose, Matrex gel blue A, and Bio-Gel A-0.5m columns. The final preparation showed one major protein band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using Bio-Gel A-0.5m filtration, the native enzyme was found to have a molecular mass of 330 kDa. With GM1 as substrate, the optimum pH of this enzyme was determined to be 5.0; the enzyme was stable between pH 4.5 and 8.5. Zn2+ at 5 mM and Cu2+, Ag+, and Hg2+ at 1 mM strongly inhibited the hydrolysis of GM1 by ceramide glycanase. The ceramide glycanase released the intact glycan chain from various glycosphingolipids in which the glycan chain is linked to the ceramide through a beta-glucosyl linkage. This enzyme also cleaved lyso-glycosphingolipids such as lyso-GM1 and lyso-LacCer and synthetic alkyl beta-lactosides. Among seven alkyl beta-lactosides tested, the enzyme only hydrolyzed the ones with an alkyl chain length of four or more carbons. The enzyme also hydrolyzed 2-(octadecylthio)ethyl O-beta-lactoside and 2-(2-carbomethoxyethylthio)ethyl O-beta-lactoside. p-Nitrophenyl, benzyl, and phytyl beta-lactosides, on the other hand, were not hydrolyzed. These results suggest that the enzyme can recognize the hydrophobic portion of glycolipid substrates. The fact that 2-(2-carbomethoxyethylthio)ethyl O-beta-N-acetyllactosaminide and DiGalCer were refractory to the enzyme indicated that in the substrate the first sugar attached to the hydrophobic chain cannot be N-acetylglucosamine and galactose. Furthermore, dodecyl maltoside, Gal alpha 1----6Glc beta Cer, and the LacCer in which the --CH2OH of the galactose was converted into --CHO were also resistant to the enzyme, and Man beta 1----4 Glc beta Cer was hydrolyzed at a much slower rate than LacCer. These results indicate that the nature and the linkage of the sugar attached to the glucose have a profound effect on the action of this enzyme. The hydrolysis of glycosphingolipids by ceramide glycanase is stimulated by bile salts. Among various bile salts tested, sodium cholate at a concentration of 1 microgram/microliter was found to be most effective in stimulating the hydrolysis of various glycosphingolipids with the exception of LacCer. For LacCer, sodium taurodeoxycholate at a concentration of 2-3 micrograms/microliters was most effective. Tween 20, Nonidet P-40, and Triton X-100 did not stimulate the hydrolysis of GM1.(ABSTRACT TRUNCATED AT 400 WORDS)     

A putative processing enzyme from Aplysia that cleaves dynorphin A at the single arginine residue

A peptidase activity cleaving at single arginine residues has been detected in extracts of the atrial gland of Aplysia Californica. The enzyme assay consisted of incubation of enzyme with the mammalian opioid peptide dynorphin A and detection by specific radioimmunoassay of dynorphin (1-8), a single arginine cleavage product. The peptidase activity was characterized following chromatography on DEAE-cellulose. Activity was abolished by a thiol-directed inhibitor and chelators and activated by dithiothreitol and cobalt chloride. The pH optimum was 6.2 in phosphate buffer. Analysis of the products of two substrates suggested that cleavage was occurring on the amino side of the arginine residue.     

Equine gene mapping: Close linkage between the loci for soluble malic enzyme and Xk (Pa)

Resolution of equine soluble malic enzyme phenotypes is greatly improved by isoelectric focusing as compared with starch gel electrophoresis. Phenotype differences can be recognized in plasma as well as haemolysates. The locus for soluble malic enzyme (ME1) is closely linked to the locus for Xk.     

HIV protease cleaves poly(A)-binding protein

The PABP [poly(A)-binding protein] is able to interact with the 3' poly(A) tail of eukaryotic mRNA, promoting its translation. Cleavage of PABP by viral proteases encoded by several picornaviruses and caliciviruses plays a role in the abrogation of cellular protein synthesis. We report that infection of MT-2 cells with HIV-1 leads to efficient proteolysis of PABP. Analysis of PABP integrity was carried out in BHK-21 (baby-hamster kidney) and COS-7 cells upon individual expression of the protease from several members of the Retroviridae family, e.g. MoMLV (Moloney murine leukaemia virus), MMTV (mouse mammary tumour virus), HTLV-I (human T-cell leukaemia virus type I), SIV (simian immunodeficiency virus), HIV-1 and HIV-2. Moreover, protease activity against PABP was tested in a HeLa-cell-free system. Only MMTV, HIV-1 and HIV-2 proteases were able to cleave PABP in the absence of other viral proteins. Purified HIV-1 and HIV-2 proteases cleave PABP1 directly at positions 237 and 477, separating the two first RNA-recognition motifs from the C-terminal domain of PABP. An additional cleavage site located at position 410 was detected for HIV-2 protease. These findings indicate that some retroviruses may share with picornaviruses and caliciviruses the capacity to proteolyse PABP.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (mlDL): Structure-function relationship

Based on the three-dimensional model of the bifunctional enzyme destabilase-lysozyme of the medicinal leech (mlDL) in complex with trimer of N-acetylglucosamine (NAG)₃ by site-directed mutagenesis method, the functional role of the group of amino acids (Glu14, Asp26, Ser29, Ser31, Lys38, His92) in manifestation of lysozyme (glycosidase, muramidase) and isopeptidase activities has been investigated by site-directed mutagenesis. The results obtained go well with hypothesis, that lysozyme active site of mlDL includes catalytic Glu14 and Asp26 residues, and isopeptidase site functions as Ser/Lys catalytic dyad presented by catalytic residues Ser29 and Lys38. Thus, among the invertebrate lysozymes, mlDL presents the first example of a bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules

A unique proenkephalin converting enzyme specifically generating enkephalin was partially purified from lysates of adrenal chromaffin granules. The enzyme, whose molecular weight is estimated as ca. 220,000, is thiol-dependent protease, with optimal pH at around 5.5. The enzyme converts proenkephalin to enkephalins by cleaving specifically at the sites of consecutive basic amino acid residues. The enzyme also converts BAM-12P, an adrenal “big” Met-enkephalin, to Met-enkephalin in a similar manner. During the enzyme reaction, formation of [Arg⁶]-Met-enkephalin was not observed. Additionally, [Arg⁶]-enkephalins were not converted to enkephalins by the enzyme. Consequently, the enzyme was proved to be a unique converting enzyme distinct from either trypsin-like or carboxypeptidase B-like proteases.     

Coordination between enzyme specificity and intracellular compartmentation in the control of protein-bound oligosaccharide biosynthesis

This laboratory has developed schemes for the control of biosynthesis of N- and O-glycosyl oligosaccharides based on studies in cell-free systems of glycosyl-transferase substrate specificities. These schemes are based on assumptions that may not be universally correct. For example, we have ignored the possible compartmentation of reactions in different cells or in different organelles within a cell. Recent evidence has indicated that the Golgi apparatus has at least three functionally distinct regions (cis, medial and trans). The addition of galactosyl and sialyl residues to the antennae of complex and hybrid N-glycans probably occurs entirely within the trans-cisternae while the N-acetylglucosaminyl-transferases which initiate these antennae appear to be located in a denser region of the Golgi (cis and/or medial cisternae). We have constructed a modified scheme for the biosynthesis of the antennae of N-glycans. This scheme combines our substrate specificity data (H. Schachter, S. Narasimhan, P. Gleeson and G. Vella, 1983, Can. J. Biochem. Cell Biol., 61, 1049-1066) with compartmentation data. It provides a basis for understanding the control of glycoprotein synthesis in normal tissues and in certain lectin-resistant mutant cell lines.     

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37°C, the Leu¹⁰-Leu¹¹ bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu¹¹-Val¹²-Tyr¹³-Ser¹⁴ peptide as products, with a specific activity of 1.35 pmol AI/min/mg (K_m 22 μM; K_cat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC₅₀ = 4.6 μM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.     

A unique enzyme from Saccharothrix sp. catalyzing d-amino acid transfer

A newly isolated actinomycete belonging to Saccharothrix sp. was found to produce a unique enzyme catalyzing d-amino acid transfer. The enzyme, which was tentatively named d-amino acid transferase, was purified 2600-fold to electrophoretic homogeneity and the molecular mass was 41 kDa. The enzyme was d-configuration specific and recognized aromatic d-amino acid esters to form oligo d-amino acid esters. d-Phenylalanine ester was favored as substrate over other d-amino acid esters. The optimum conditions for oligo d-phenylalanine ester formation by d-amino acid transferase were pH 7.0 and 40°C. The enzyme was inhibited by DAN, EPNP and DFP.     

Genetic linkage relationships between two enzyme loci,Est-2 and acph,in the mosquito Aedes (Finlaya) togoi

An esterase locus (Est-2), coding for carboxylesterase, and an acid phosphatase locus (Acph) were genetically studied by agar gel electrophoresis in the mosquito Aedes (Finlaya) togoi. The Est-2 and Acph variants occur as a monomer and a dimer, respectively. Both enzyme loci are linked to the sex locus (M) and s (straw-colored larva); the gene arrangement and recombination distances were Est-2—12.6%—s—31.7%—M—2.9%—Acph—3.2%—Est-3. The Est-3 locus was previously shown to code for carboxylesterase.This work was supported by Grant AI 16983-02 from the National Institutes of Health, Bethesda, Md.     

An enzymatic activity in uninfected cells that cleaves the linkage between poliovirion RNA and the 5' terminal protein.

The 5' terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of heLa cells and is activated by Mg++. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5' terminal protein. The role of this enzyme in the uninfected cell is not known.     

Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity

Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene.     

Determination of the glycosaminoglycan-protein linkage region oligosaccharide structures of proteoglycans from Drosophila melanogaster and Caenorhabditis elegans

Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl- or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)- for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution.