Subcellular fractionation of polyprenyl diphosphate synthase activities responsible for the syntheses of polyprenols and dolichols in spinach leaves

Polyisoprenoid alcohols occurring in spinach leaves were analyzed by a two-plate TLC method. Z,E-mixed polyprenols (C(55-60)), glycinoprenols (C(50-55)), and solanesol (C(45)) were mainly found in chloroplasts, whereas dolichols (C(70-80)) were mainly found in microsomes. Analysis of enzymatic products derived from [1-(14)C]isopentenyl diphosphate and farnesyl diphosphate (FPP) with subcellular fractions revealed that chloroplasts and microsomes had the ability to synthesize Z,E-mixed polyprenyl (C(50-65)) and all E-polyprenyl (C(45-50)) diphosphates, and Z,E-mixed polyprenyl (C(70-85)) diphosphates, respectively. FPP and geranylgeranyl diphosphate (GGPP) were both accepted for these enzymatic reactions, the former being a better substrate than the latter. NMR analysis of naturally occurring spinach Z,E-mixed polyprenol (C(55)) and dolichol (C(75)) revealed that the number of internal trans isoprene residues in the former was three in comparison with two internal trans residues found for the latter. These results indicate that two kinds of polyprenyl diphosphate synthases occur in spinach: One is the chloroplast enzyme involved in the synthesis of the shorter-chain (C(50-65)) Z,E-mixed polyprenols and the other is the microsomal enzyme involved in the synthesis of longer-chain (C(70-85)) Z,E-mixed polyprenols, which is converted to dolichols.     

Interplay between the cis-prenyltransferases and polyprenol reductase in the yeast Saccharomyces cerevisiae

Dolichol formation is examined in three Saccharomyces cerevisiae strains with mutations in the ERG20 gene encoding farnesyl diphosphate synthase (mevalonic acid pathway) and/or the ERG9 gene encoding squalene synthase (sterol synthesis pathway) differing in the amount and chain length of the polyisoprenoids synthesized. Our results suggest that the activities of two yeast cis-prenyltransferases Rer2p and Srt1p and polyprenol reductase are not co-regulated and that reductase may be the rate-limiting enzyme in dolichol synthesis if the amount of polyisoprenoids synthesized exceeds a certain level. We demonstrate that reductase preferentially acts on typical polyprenols with 13-18 isoprene residues but can reduce much longer polyprenols with even 32 isoprene residues.     

Mutants in dolichol synthesis: conversion of polyprenol to dolichol appears to be a rate-limiting step in dolichol synthesis

Chinese hamster ovary (CHO) cells of the Lec9 recessive complementationgroup display a distinctive profile of resistance to a varietyof toxic lectins. In addition, they accumulate cis--unsaturatedpolyprenol and use mainly polyprenol rather than dolichol tosynthesize the glycosylated lipids used in asparagine-linkedglycosylation of proteins. The primary defect in these cellsis thought to result from a deficiency in polyprenol reductaseactivity. Three new mutants were isolated and determined tohave qualitatively, although not quantitatively, similar lectinresistance profiles to Lec9 cells. Two of these mutants (Abr^Rand Ric^R) also contained polyprenol rather than dolichol. Thelectin resistance profile of an independent mutant which accumulatespolyprenol, F2A8, was also found to be qualitatively similarto the Lec9 pattern. The relationship among these mutants wasanalysed in more detail by construction of cell—cell hybrids.Lectin resistance profiles of the hybrids demonstrated thatAbr^R, Ric^R and F2A8 fell into the Lec9 complementation group.Analysis of prenols in the hybrids also showed that F2A8 wasa member of the Lec9 group. Surprisingly, a significant fractionof the prenols found in Lec9 Parent hybrids was polyprenol(up to 30% of the neutral fraction), whereas the prenols foundin Parent Parent hybrids were nearly exclusively dolichol(97% of the neutral lipid fraction). Therefore, reduction ofpolyprenol to dolichol appears to be a rate-limiting step inthe synthesis of dolichol since hybrids with differing numbersof wild-type alleles can be biochemically distinguished. CHO cells dolichol lectins mutants polyprenol reductase     

Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings.

Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein.h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein.h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds.     

Separation and purification of dolichol and dolichyl phosphate by anion-exchange paper chromatography: application to cultured cells.

Dolichyl phosphate, dolichol C80-105 (dolichol 17:dihydroheptadecaprenol-dolichol 21:dihydrohexeicosaprenol), and dolichol C55 (dolichol 11:dihydroundecaprenol) were separated by anion-exchange paper chromatography. Squalene, sterols, phospholipids, anionic glycolipids, and glycerol did not migrate as dolichyl phosphate, dolichol C80-105, and dolichol C55 under our elution conditions. However, since the Rf of triglycerides was similar to that of dolichol C80-105, saponification, prior to chromatography, removed traces of triglycerides. Silica gel thin-layer chromatography (TLC) allowed the separation of dolichol C80-105 from dolichol C55, whereas dolichyl phosphate was eluted with other lipids. Incubation of spontaneously transformed cells derived from rat astrocytes primary cultures with [2-14C]acetate, saponification of the extracted lipids, and anion-exchange paper chromatography revealed the presence of radioactive dolichyl phosphate and dolichol C80-105 (15 pmol/mg protein). Extraction of labeled dolichyl phosphate followed by acid phosphatase treatment and subsequent analysis on TLC confirmed the identity of dolichyl phosphate since all the radioactivity was associated with dolichol C55. Treatment of the transformed cells with 30 microM 7-ketocholesterol or 7 beta-hydroxycholesterol stimulated markedly (two- to threefold) the incorporation of [2-14C]-acetate in both dolichol C80-105 and dolichyl phosphate. These data demonstrate that anion-exchange paper chromatography is technically suitable for the separation and analysis of dolichol C55, dolichol C80-105, and dolichyl phosphate in cultured cells prelabeled with radioactive precursors.     

Lec9 CHO glycosylation mutants are defective in the synthesis of dolichol

Lec9 Chinese hamster ovary cells were found previously to be defective in the synthesis of N-linked glycans. This appeared to be the result of a defect in the synthesis of oligosaccharide lipid and lipid phosphate (Rosenwald, Stanley, and Krag. 1989. Mol. Cell. Biol. 9: 914-924). In this study we analyzed the steady state levels of long-chain polyisoprenyl lipids in Lec9 cells. We found that Lec9 cells are defective in the synthesis of dolichol. They accumulated a presumed precursor to dolichol, cis-a-unsaturated polyprenol and used this lipid in the synthesis of oligosaccharide lipid. Chain lengths of the activated polyprenols in Lec9 were the same lengths as dolichols in parental cells. Lec9 cells had increased levels of monosaccharylphosphoryl lipid and decreased levels of oligosaccharylpyrophosphoryl lipid compared to parental cells. The defect in Lec9 cells was specific for dolichol synthesis, since other aspects of [3H]mevalonate metabolism in Lec9 cells were the same as in parental cells. We hypothesize that Lec9 cells are defective in polyprenol reductase activity.     

Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.     

Distribution, metabolism and function of dolichol and polyprenols

Polyisoprenoid alcohols consisting of 9 or more isoprene units are present in all living cells. They can be fully unsaturated (polyprenols) or alpha-saturated (dolichol). Dolichol forms may have additional saturation at or near the omega-end. Some species contain ony dolichol or only polyprenols while others have nearly equal amounts of both types. Some polyisoprenoid alcohols consist entirely of trans isoprene units but most, including dolichol, contain both trans and cis units. Considerable advances in lipid methodology have occurred since the first review of polyisoprenoid alcohols by Hemming in 1974. For example, direct analysis of both dolichol and Dol-P by HPLC has replaced earlier methods which were often both insensitive and inaccurate. The availability of radiolabeled dolichol and polyprenols has facilitated studies concerning the metabolism and distribution of these compounds. Those studies suggest that only a small portion of the dolichol present in cells is likely to be involved in glycosylation. Polyisoprenoid alcohols are usually present at a family of homologues where each differs in size by one isoprene unit. Little or no size related specificity has been observed for any reaction involving dolichol or polyisoprenol intermediates. The overall length of polyisoprenoid alcohols may, however, affect the manner in which these compounds influence the physical and biochemical properties of membranes. Studies on the biosynthetic pathway leading from cis, trans Pol-PP by phosphatase action. The formation of the dolichol backbone from a polyprenol requires the action of an additional enzyme, an alpha-saturase. This enzyme does not always act at the level of a single common substrate, since Pol-PP, Pol-P, and polyprenol all appear to be utilized as substrates. The major product of the de novo pathway differs among different species. Dol-P would appear to be the most energy efficient end-product since it can participate directly in glycoprotein formation. Most often, however, Dol-P is not the major product of metabolic labeling experiments. In some cases, dolichol is formed so that rephosphorylation is required to provide Dol-P for participation in glycoprotein formation. The kinase responsible for this phosphorylation appears to bypass the considerable stores of dolichol present in tissues (i.e. sea urchin eggs) in favor of dolichol derived directly from de novo synthesis. Although HMGR is a major regulatory component of the pathway leading to polyisoprenoid alcohols and cholesterol, control is most often not co-ordinated.(ABSTRACT TRUNCATED AT 400 WORDS)     

The thermoacidophilic archaeon Sulfolobus acidocaldarius contains an unusually short, highly reduced dolichyl phosphate

Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.     

Involvement of sterol carrier protein-2 in dolichol biosynthesis.

The effect of sterol carrier protein-2 (SCP-2) on dolichol biosynthesis by rat liver microsomes was investigated. cis-Prenyltransferase activity was stimulated 7-fold in the presence of 5 micrograms of purified SCP-2/mg of microsomal protein, which was similar to the increase obtained by adding detergent. The polyisoprenoid pattern obtained in the presence of SCP-2 was the same as that present in rat liver, in contrast to the pattern appearing upon incubation of microsomes with detergent, which gave shorter polyisoprenoids. Like SCP-2, the cytosolic fraction from rat liver also stimulated cis-prenyltransferase. Incubation with cytosol pretreated with anti-SCP-2 showed no stimulatory effect and led to the accumulation of shorter polyisoprenoids. SCP-2 had no appreciable effect on polyprenol alpha-saturase, dolichol kinase, dolichyl phosphate phosphatase, or acyl-CoA:dolichol acyltransferase. The results demonstrate that SCP-2 greatly stimulates and may regulate the condensation reactions mediated by cis-prenyltransferase in the process of dolichol biosynthesis and permits polymerization of the polyisoprenoid to its natural chain length.     

Dolichol biosynthesis and its effects on the unfolded protein response and abiotic stress resistance in Arabidopsis

Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C(80) in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by approximately 85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.     

Properties of brain dolichol kinase activity solubilized with a zwitterionic detergent

Dolichol kinase activity is effectively solubilized by extracting calf brain microsomes with 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized kinase catalyzes the enzymatic phosphorylation of dolichols with either CTP or dCTP serving as phosphoryl donor in the presence of Ca2+. Similar Km values were calculated for CTP (7.7 microM) and dCTP (9.1 microM). Dolichol phosphorylation was inhibited by CDP and dCDP, but not CMP, ADP, GDP, or UDP. A kinetic analysis of the inhibitory effect of CDP revealed a pattern characteristic of competitive inhibition. Dolichol kinase activity was markedly stimulated by the addition of R-dolichol (C95) or S-dolichol(C95). The apparent Km value for R-dolichol(C95) and S-dolichol(C95) was 9 microM, but the Vmax for the phosphorylation reaction was 40% higher with S-dolichol(C95). Incubation of the CHAPS extract with [gamma-32P]CTP and exogenous undecaprenol(C55) resulted in the enzymatic synthesis of a radiolabeled product that was mild acid-labile and chromatographically identical to undecaprenyl monophosphate. An enzymatic comparison with a variety of polyprenol substrates indicates that the solubilized kinase prefers long-chain (C90-95) polyprenols with saturated alpha-isoprene units. The effect of exogenous phosphoglycerides on the kinase activity in the dialyzed CHAPS extracts has also been evaluated. These studies describe the properties and polyprenol specificity of stable, solubilized preparations of dolichol kinase that should be useful for further purification of the enzyme.     

Identification and characterization of a cDNA encoding a long-chain cis-isoprenyltranferase involved in dolichyl monophosphate biosynthesis in the ER of brain cells

A long-chain cis-isoprenyltransferase (cis-IPTase) located in the endoplasmic reticulum (ER) catalyzes the chain elongation stage in the pathway for the de novo biosynthesis of dolichyl monophosphate (Dol-P) in eukaryotic cells. In Saccharomyces cerevisiae, the ER-associated cis-IPTase is encoded by the RER2 gene. Mutations in the RER2 gene result in defects in growth and protein N-glycosylation. In this study a cDNA isolated from human brain (Accession No. AK023164.1), which has substantial homology to cis-IPTases from bacteria, Arabidopsis, and S. cerevisiae, has been shown to: (1) complement the growth defect; (2) restore cis-IPTase activity; dolichol and Dol-P synthesis; and (3) restore normal N-glycosylation of carboxypeptidase Y (CPY) in the yeast rer2Delta mutant. Consistent with a role in Dol-P biosynthesis, overexpression of the human cis-isoprenyltransferase (hCIT) cDNA also suppresses the temperature-sensitive growth and CPY hypoglycosylation phenotypes in sec59-1 cells which are defective in Dol-P biosynthesis due to a temperature-sensitive mutation in dolichol kinase. Overexpression of hCIT in Chinese hamster ovary (CHO) cells results in a modest increase in cis-IPTase activity associated with microsomal fractions and the appearance of a new 38kDa polypeptide that co-localizes with calnexin in the ER, the site of Dol-P biosynthesis, even though no transmembrane domains are predicted by a hydropathy plot.     

POLYPRENOL REDUCTASE2 Deficiency Is Lethal in Arabidopsis Due to Male Sterility

Dolichol is a required cofactor for protein glycosylation, the most common posttranslational modification modulating the stability and biological activity of proteins in all eukaryotic cells. We have identified and characterized two genes, PPRD1 and -2, which are orthologous to human SRD5A3 (steroid 5α reductase type 3) and encode polyprenol reductases responsible for conversion of polyprenol to dolichol in Arabidopsis thaliana. PPRD1 and -2 play dedicated roles in plant metabolism. PPRD2 is essential for plant viability; its deficiency results in aberrant development of the male gametophyte and sporophyte. Impaired protein glycosylation seems to be the major factor underlying these defects although disturbances in other cellular dolichol-dependent processes could also contribute. Shortage of dolichol in PPRD2-deficient cells is partially rescued by PPRD1 overexpression or by supplementation with dolichol. The latter has been discussed as a method to compensate for deficiency in protein glycosylation. Supplementation of the human diet with dolichol-enriched plant tissues could allow new therapeutic interventions in glycosylation disorders. This identification of PPRD1 and -2 elucidates the factors mediating the key step of the dolichol cycle in plant cells which makes manipulation of dolichol content in plant tissues feasible.     

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

Biosynthesis of the wall teichoic acid in Bacillus licheniformis

1. The biosynthesis of the wall teichoic acid, poly(glycerol phosphate glucose), has been studied with a particulate membrane preparation from Bacillus licheniformis A.T.C.C. 9945. The precursor CDP-glycerol supplies glycerol phosphate residues, whereas UDP-glucose supplies only glucose to the repeating structure of the polymer. 2. Synthesis proceeds through polyprenol phosphate derivatives, and chemical studies and pulse-labelling techniques show that the first intermediate is the phosphodiester, glucose polyprenol monophosphate. CDP-glycerol donates a glycerol phosphate residue to this to give a second intermediate, (glycerol phosphate glucose phosphate) polyprenol. 3. The glucose residue in the lipid intermediates has the beta configuration, and chain extension in the synthesis of polymer occurs by transglycosylation with inversion of anomeric configuration at two stages.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.     

In vivo biosynthesis of the saturated isoprene unit of dolichyl phosphate

Reduction of the e-isoprene unit of polyprenols to form dolichols was studied in vivo using³H-polyprenol derivatives as substrates and liposomes as carriers. Liposomes containing labeled polyprenol, polyprenyl phosphate, or polyprenyl pyrophosphate were injected through the portal vein into the livers of rats under anesthesia. Uptake and conversion of the labeled compounds to dolichol derivatives was studied at different intervals. The greatest conversion to dolichol derivatives was found with polyprenyl pyrophosphate and polyprenyl monophosphate, with 31% and 8% of the absorbed dose converted respectively. Less than 0.2% of the absorbed polyprenol was converted to dolichol derivatives. These results suggest that the substrate for the -isoprene reductase involved in dolichol biosynthesis is either polyprenyl monophosphate or polyprenyl pyrophosphate, or both.     

Structure-function analysis of the human integrin VLA-4 (alpha 4/beta 1). Correlation of proteolytic alpha 4 peptides with alpha 4 epitopes and sites of ligand interaction.

The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti-alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.     

Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes

Previously mutations in a putative protein O -mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, φC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt ⁻ derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor . A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C₄₅-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo .