The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147+/-24.1 nM and 52+/-9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6+/-0.5 to 0. 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8+/-0.04 nm, corresponding to a hydrodynamic volume of 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55x10(-24) m3, suggesting that haem availability regulates eNOS dimerization.

The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

Recently, we reported a novel congenital disorder of glycosylation (CDG-IIb) caused by severe deficiency of the glucosidase I. The enzyme cleaves the alpha1,2-glucose residue from the asparagine-linked Glc(3)-Man(9)-GlcNAc(2) precursor, which is crucial for oligosaccharide maturation. The patient suffering from this disease was compound-heterozygous for two mutations in the glucosidase I gene, a T-->C transition in the paternal allele and a G-->C transition in the maternal allele. This gives rise in the glucosidase I polypeptide to the substitution of Arg486 by Thr and Phe652 by Leu, respectively. Kinetic studies using detergent extracts from cultured fibroblasts showed that the glucosidase I activity in the patient's cells was < 1% of the control level, with intermediate values in the parental cells. No significant differences in the activities of other processing enzymes, including oligosaccharyltransferase, glucosidase II, and Man(9)-mannosidase, were observed. By contrast, the patient's fibroblasts displayed a two- to threefold higher endo-alpha1,2-mannosidase activity, associated with an increased level of enzyme-specific mRNA-transcripts. This points to the lack of glucosidase I activity being compensated for, to some extent, by increase in the activity of the pathway involving endo-alpha1,2-mannosidase; this would also explain the marked urinary excretion of Glc(3)-Man. Comparative analysis of [(3)H]mannose-labeled N-glycoproteins showed that, despite the dramatically reduced glucosidase I activity, the bulk of the N-linked carbohydrate chains (>80%) in the patient's fibroblasts appeared to have been processed correctly, with only approximately 16% of the N-glycans being arrested at the Glc(3)-Man(9-7)-GlcNAc(2) stage. These structural and enzymatic data provide a reasonable basis for the observation that the sialotransferrin pattern, which frequently depends on the type of glycosylation disorder, appears to be normal in the patient. The human glucosidase I gene contains four exons separated by three introns with exon-4 encoding for the large 64-kDa catalytic domain of the enzyme. The two base mutations giving rise to substitution of Arg486 by Thr and Phe652 by Leu both reside in exon-4, consistent with their deleterious effect on enzyme activity. Incorporation of either mutation into wild-type glucosidase I resulted in the overexpression of enzyme mutants in COS 1 cells displaying no measurable catalytic activity. The Phe652Leu but not the Arg486Thr protein mutant showed a weak binding to a glucosidase I-specific affinity resin, indicating that the two amino acids affect polypeptide folding and active site formation differently.

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774^777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC SH for nNOS was 18 þ 6 W WM GST-PIN with 63 nM nNOS after 30 min at 37³C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K w for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.z 1998 Federation of European Biochemical Societies.

Man,-mannosidase, a processing enzyme found in the endoplasmic reticulum (ER), catalyses the removal of three distinct mannose residues from peptide-bound Man,-GlcNAc, oligosaccharides producing a single Man, isomer [Bause, E., Breuer, W., Schweden, J., Roesser, R. & Geyer, R. (1992) Eur: J. Biochem. 208,. We have isolated four Man,-mannosidase-specific clones from a human kidney cDNA library and used these to construct a full-length cDNA of 3250 base pairs. A single open reading frame of 1875 nucleotides encodes a protein of approximately 71 kDa, consistent with data from immunological studies. Analysis of the coding sequence predicts that Man,-mannosidase is a type II transmembrane protein consisting of a short cytoplasmic polypeptide tail, a single transmembrane domain acting as a non-cleavable signal sequence and a large luminal catalytic domain. This domain architecture closely resembles that of other ER and Golgi-located processing enzymes, pointing to common structural motifs involved in membrane insertion and topology. The protein sequence of the Man,-mannosidase contains three potential N-glycosylation sites of which only one site is used. The amino acid sequence of several peptide regions, including a calcium-binding consensus sequence, bears striking similarities to an ER a-l,2-mannosidase from yeast, whereas, by contrast, no sequence similarity was detectable with rat liver ER a-mannosidase and Golgi a-mannosidase II. This finding may indicate that the mammalian a-mannosidases, which differ significantly in their substrate specificity, are coded for by evolutionarily unrelated genes, providing an attractive means of regulation and fine-tuning oligosaccharide processing, not only at the enzymic but also at the transcriptional level.Man,-mannosidase, a transmembrane protein, found in the endoplasmic reticulum (ER), is one of at least three different exo-a1,2-mannosidases which have been implicated as being involved in the early processing pathway of mammalian N-glycoproteins [I -51. A stable and catalytically active 49-kDa fragment of the enzyme has been purified recently from pig liver crude microsomes and found to degrade selectively free and peptide-bound Man,-GlcNAc, oligosaccharides to a single Man,-isomer [6, 71. This unique substrate specificity in particular distinguishes Man,-mannosidase from a second ER al,2-mannosidase and from Golgi mannosidase I, which both act on the same Man,-GlcNAc, precursor but generate oligosaccharide intermediates of different size and structure [3, 81. In addition to these a1,2-specific exoenzymes, an endo-al,2-mannosidase activity which removes Glc,-,-Man units from previously glucosylated oligosaccharides, has been identified recently in rat liver [9, 101. The biological role of these al,2-mannosidases is still unCorrespondence to E. Bause,

Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.

Man,-mannosidase, one of three different al,2-exo-mannosidases known to be involved in N-linked oligosaccharide processing, has been cloned in AgtlO, using a mixed-primed pig liver cDNA library. Three clones were isolated which allowed the reconstruction of a 2731-bp full-length cDNA. The cDNA construct contained a single open reading frame of 1977 bp, encoding a 659-residue polypeptide with a molecular mass of =73 kDa. The Man,-mannosidase specificity of the cDNA construct was verified by the observation that all peptide sequences derived from a previously purified, catalytically active 49-kDa fragment were found within the coding region. The N-terminus of the 49-kDa fragment aligns with amino acid 175 of the translated cDNA, indicating that the catalytic activity is associated with the C-terminus.Transfection of COS 1 cells with the Man,-mannosidase cDNA gave rise to a > 30-fold over-expression of a 73-kDa protein whose catalytic properties, including substrate specificity, susceptibility towards amannosidase inhibitors and metal ion requirements, were similar to those of the 49-kDa enzyme fragment. Thus deletion of 174 N-terminal amino acids in the 73-kDa protein appears to have only marginal influence on the catalytic properties.Structural and hydrophobicity analysis of the coding region, as well as the results from tryptic degradation studies, point to pig liver Man,-mannosidase being a non-glycosylated type-I1 transmembrane protein. This protein contains a 48-residue cytosolic tail followed by a 22-residue membrane anchor (which probably functions as internal and non-cleavable signal sequence), a lumenal = 100-residue-stem region and a large 49-kDa C-terminal catalytic domain. As shown by immuno-fluorescence microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the endoplasmic reticulum, in contrast to COS 1 Man,-mannosidase from human kidney which is Golgi-located [Bieberich, E. & Bause, E. (1995) E m J. Biochem. 233, 644-6491. Localization of the porcine enzyme in the endoplasmic reticulum is consistent with immuno-electron-microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man,-mannosidase is, therefore, enzyme-specific rather than a COS-1-cell-typical phenomenon. Since we oberserve = 81 % sequence similarity between the two amannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence-dependent.Keywords: Man,-mannosidase; cDNA cloning; COS 1 cell expression; type I1 membrane protein; endoplasmic reticulum localization.Re-modelling of the asparagine-linked Glc,-Man,-GlcNAc, precursor to give the mature high-mannose or complex-type sugar chain, takes place through a complex sequence of reactions involving a-glycosidases and glycosyltransferases [ 1, 21. Processing begins with the removal of three glucose residues by glucosidase I and 11, followed by cleavage of four al,2-linked mannose residues from Man,-GlcNAc,. The resulting Man,-GlcNAc, intermediate may then be modified...

The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S5 and Table S1.