The storage products in genetic and swainsonine-induced human mannosidosis.

Swainsonine induces the accumulation of mannose-rich oligosaccharides in human fibroblasts. The composition of the storage products shows that swainsonine completely inhibits lysosomal alpha-D-mannosidase and alters processing of glycoproteins by inhibiting Golgi alpha-D-mannosidase II. Comparison of the storage products in genetic and swainsonine-induced mannosidosis suggests that human fibroblasts contain a lysosomal alpha-D-mannosidase that is unaffected in genetic mannosidosis.     

The nature of the residual alpha-mannosidase in plasma in bovine mannosidosis.

Acidic alpha-mannosidase (EC 3.2.1.24), optimum pH 4.25, is absent from the plasma of Angus calves with mannosidosis, and the residual alpha-mannosidase activity has an optimum pH of 5.5, intermediate between that of the acidic and neutral alpha-mannosidases. This 'intermediate' alpha-mannosidase differs from the acidic form in its kinetic properties, its lack of marked inhibition by EDTA and its thermolability at 55 degrees C and physiological pH. Isoelectric focusing and ion-exchange chromatography show that it exists in at least two forms. The presence of a secondary peak at pH 5.5 in the pH/activity profile of normal plasma and the effect of heating at 55 degrees C indicate that such a form is present in normal plasma. The residual activity in the plasma of a calf with mannosidosis is therefore probably not the product of the defective gene. A differential assay, based on their different stabilities at 55 degrees C, has been developed for measuring the acidic and intermediate alpha-mannosidases in plasma. There was no correlation between the concentrations of the two enzymes in the plasma of Angus cows heterozygous for mannosidosis or in the plasma of normal animals. This precludes the use of the intermediate form as a reference enzyme for the acidic activity in a test for heterozygosity for mannosidosis based on the gene-dosage phenomenon. The concentrations of the intermediate activity were comparable in normal animals and animals homozygous or heterozygous for mannosidosis.     

人及哺乳动物受精与糖蛋白的关系

越来越多的研究表明精卵识别与精子质膜及卵透明带中所含糖蛋白有直接关系。在精卵识别中存在着精子受体和卵透明带（zona pellucida,ZP）配体相互作用的糖类识别机制及精子质膜与卵子质膜的糖蛋白识别。本文主要从结构功能上对与受精相关的精卵表面糖蛋白作一介绍。卵子表面受精相关的糖蛋白主要是ZP1、ZP2、ZP3。与卵子表面糖蛋白相比,精子表面参与精卵识别的糖蛋白种类较多,在SP56、SP95、PH－20、FA－1、甘露糖结合蛋白、顶体素、fertilin蛋白等。     

Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

The catabolism of plasma glycoproteins in normal and injured rats

The catabolism of (14)C-labelled plasma glycoprotein in rats was studied after injecting homologous plasma protein labelled in the N-acetylglucosamine and sialic acid moieties. In normal animals the catabolism was approximately described by a four-compartment model. The fractional rate of catabolism of the plasma-protein amino sugar was found to be 0.0305hr.(-1), corresponding to the degradation of 2.75mumoles/hr. The (14)C label was eliminated from the animals largely as carbon dioxide with a small proportion appearing in the urine. Freely circulating amino sugars or glycopeptides did not appear in the plasma as a result of the catabolic processes, and there was no evidence that the protein-bound amino sugars were reutilized in biosynthetic processes. A study of the distribution of (14)C label in the carcasses of animals 24hr. after injection provided evidence that the gastrointestinal tract accounted for 25-38% of the total catabolic pool; the lungs, kidneys, spleen and liver also appeared to contribute to catabolism. Studies were conducted with rats that had been treated with turpentine to induce an inflammatory reaction; the results could not be analysed kinetically, since the metabolism of plasma proteins in these animals did not appear to be in a steady state. The injected plasma protein disappeared from the intravascular pool more quickly than in normal animals, but there were no significant differences in the rates of excretion of the (14)C label.     

Comparison of rat and human major platelet glycoproteins.

1. Using electrophoretic techniques combined with various detection methods we ascribed rat platelet glycoproteins (GPs) related to human GPIb, GPIIb and GPIIIa. 2. Rat GPIIb and GPIIIa crossreacted with rabbit polyclonal antibodies against human GPIIb and GPIIIa. 3. Species differences in glycosylation of GPs were shown using various lectins. 4. Molecular mass of rat major GPs was determined by SDS-PAGE (unreduced, reduced, kDa): GPIb (200, 166/26), GPIIb (140, 120/32) and GPIIIa (96, 106). 5. Isoelectric points of rat GPIIb and GPIIIa are shifted to the alkaline region as compared to human related GPs.     

Comparison of human and bovine protoporphyria.

Protoporphyria (PP) is an inherited disorder of porphyrin metabolism in man in which there is excessive accumulation and excretion of protoporphyrin. Recently, a similar disorder has been described in cattle. In this report, the clinical, biochemical, and genetic features of bovine and human PP are compared. Human and bovine PP are characterized by photosensitivity and elevation of erythrocyte and fecal protoporphyrin levels. In both disorders, a deficiency of heme synthase activity is present in all tissues which have been examined. The diseases differ clinically in that hepatobiliary disease has been found thus far only in human PP. They also have different inheritance patterns. Human PP is an autosomal dominant disease, while initial studies strongly suggest that there is an autosomal recessive pattern of inheritance in bovine PP.     

Terminal glycosylation of bovine uroplakin III, one of the major integral-membrane glycoproteins of mammalian bladder

Uroplakin III (UPIII) is one of the major transmembrane glycoproteins exposed at the luminal face of mammalian bladder. We investigated the terminal glycosylation of bovine UPIII in order to ascertain whether it contains the alpha 2,3-sialylated sequence thus potentially serving as a receptor for uropathogenic Escherichia coli expressing type S adhesins. We report the occurrence of sialic acid in alpha 2,3- and alpha 2,6-linkage to galactose in bovine UPIII glycans as evidenced by the sensitivity of UPIII to both Vibrio cholera and Newcastle disease virus neuraminidase and by the colocalization of UPIII antigen and material detected by lectins of Sambucus nigra and Maackia amurensis on the luminal face of the bladder. We also present evidence that UPIII glycans are capped by Gal-alpha 1,3-Gal epitope. Consistently, alpha 2,3- and alpha 2, 6-sialyltransferase, as well as alpha 1,3-galactosyltransferase were found to be present in the cells detached from the luminal side of bovine bladder, which are responsible for the UPIII biosynthesis. The putative role of UPIII sialylated glycans in enhancing the uropathogenicity of E. coli expressing type S adhesins is discussed.     

Functional differences in the peplomer glycoproteins of feline coronavirus isolates.

The feline coronaviruses can be divided into two distinct antigenic groups on the basis of antigenic differences found on the peplomer (E2) glycoprotein of the virus. Because the E2 glycoprotein is responsible for many of the biological functions of coronaviruses, experiments were done to determine whether there were any E2 functional differences between these two antigenic groups. The avirulent feline infectious peritonitis virus (FIPV) isolate FIPV-UCD-2, which has one antigenic type of E2, was less rapidly internalized and could not spread from cell to cell in the presence of neutralizing antibody. Two virulent isolates, FIPV-DF2 and FIPV-79-1146, as well as the non-FIP-causing feline enteric coronavirus (FECV) isolate FECV-79-1683, all of which have the second antigenic type of E2, were very rapidly internalized and were able to spread from cell to cell despite the presence of neutralizing antibody. The avirulent FIPV-UCD-2 and FECV-79-1683 isolates were more labile at 37 degrees C at pHs of 6.5 and above than were the virulent FIPV-DF2 and FIPV-79-1146 isolates.     

Comparison of glycoproteins from ten human Mycoplasma species.

Multiple bands of glycoprotein, rare in procaryotes, were detected in ten human Mycoplasma species by staining with periodic acid-Schiff (PAS) reagent after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A major contaminant formed in Hayflick medium (H medium), corresponding to an apparent molecular weight of about 80 kD, was eliminated by using the organisms grown in PPLO broth supplemented with PPLO serum fraction (P medium), except that M. genitalium and M. pneumoniae were grown in H medium as monolayers on the glass surface. The comparison of glycoproteins among ten human Mycoplasma species indicated that their profiles were shown to be species-specific. However, those of M. buccale and M. faucium were very similar, and M. pneumoniae and M. genitalium seemed to be related.     

Sequence of the bovine CD18-encoding cDNA: comparison with the human and murine glycoproteins.

The bovine cDNA (CD18) encoding CD18, a cell-surface glycoprotein involved in multiple leukocyte functions, was sequenced and compared with the human and murine sequences. Portions of the 5'- and 3'-untranslated regions of the nucleotide sequences are conserved among the three species, including a 3' A+T-rich region believed to regulate mRNA stability and translational efficiency. The 2833-bp bovine sequence coded for a protein of 769 amino acids (aa). Overall, the deduced aa sequences were greater than 80% identical among the three species. The aa 96-389 and those in the cytoplasmic domain were very highly conserved with approx. 95% aa identity. All Cys residues and potential Asn-glycosylation sites present in the bovine sequence were also present in the human and murine sequences. The aa identity was also found in those regions where mutations were found to cause the genetic disease, leukocyte adhesion deficiency. These data identify functionally important regions of the CD18 mRNA and protein.     

B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.     

High proportion of mannosidosis and fucosidosis among lysosomal storage diseases in Cuba

Although lysosomal storage disorders (LSDs) are considered individually rare, as a group they present a non-negligible frequency. Few studies have been made of populational occurrence of LSDs; they have been conducted predominantly on Caucasian populations. We studied the occurrence of LSDs in Cuba. Data from individuals who had been referred to the Institute of Neurology and Neurosurgery in Havana from hospitals all over the country between January 1990 and December 2005 were analyzed. This institute was the only laboratory to provide enzyme-based diagnostic testing for 19 LSDs in Cuba during this period. Occurrence rates were calculated by dividing the number of postnatal diagnoses by the number of births during the study period. The combined occurrence of LSDs in Cuba was 5.6 per 100,000, lower than that reported in other studies conducted on Caucasian populations. The most frequent individual LSDs were: mucopolysaccharidosis type I (1.01 per 100,000) and, surprisingly, alpha-mannosidosis (0.72 per 100,000) and fucosidosis (0.62 per 100,000). These findings may be related to specific genetic characteristics and admixture of the Cuban population. This is the first comprehensive study of the occurrence of LSDs in Cuba. We conclude that the epidemiology of these diseases can vary regionally, and we stress the need for similar surveys in other Latin American countries.     

Drug-induced lysosomal storage of sulfated glycosaminoglycans in cultured bovine and human fibroblasts.

Several di-cationic amphiphilic compounds are known to cause lysosomal accumulation of sulfated glycosaminoglycans (sGAG) in intact rats and in cultured rat fibroblasts. The purpose of the present investigation was to examine whether this drug side effect also occurs in bovine and human cells. Cultured fibroblasts from both species were exposed to tilorone (3 microM and 5 microM) for 72 h; lysosomal sGAG-storage was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The cytological alterations as well as the radiochemical results in both species were in good agreement with previous data from rat fibroblasts. The present findings indicate that the drug-induced lysosomal storage of sGAG is a species-independent phenomenon. Thus, cultured bovine and human fibroblasts are a suitable model for further studies concerning the as yet unknown molecular mechanisms underlying this adverse drug action.     

Residual mannosidase activity in human mannosidosis: characterization of the mutant enzyme.

The prenatal diagnosis of affected fetuses in two families at risk for mannosidosis gave us the opportunity to study the residual alpha-mannosidase activity. We found an altered acidic alpha-mannosidase characterized by lowered affinity toward the substrate, displacement of maximal activity toward pH 4-5, thermal lability, different migration in electrophoresis, and apparent change in molecular weight at alkaline pHs. The immunological properties seem unchanged since the enzyme was precipitated by an antiacidic alpha-mannosidase antiserum. The mutant enzyme instability, provoked by dialysis, and its reactivation after addition of dialysis fluid, suggests an association-dissociation phenomenon. We propose a possible hypothesis that a low molecular weight ligand is necessary to maintain the activity of the mutant enzyme.     

Origin of oestrus-associated glycoproteins in bovine oviductal fluid.

The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi [35S]methionine in serum-free medium for 6 h at 37 degrees C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.