Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]     

The genus-specific lipopolysaccharide epitope of Chlamydia is assembled in C. psittaci and C. trachomatis by glycosyltransferases of low homology

Chlamydiae possess a genus-specific epitope that is located on the lipopolysaccharide (LPS) and is composed of a 3-deoxy-d -manno-octulosonic acid (Kdo) trisaccharide of the sequence αKdo-(2→8)–αKdo–(2→4)-αKdo. In Chlamydia trachomatis, this trisaccharide is biosynthetically generated through the action of a multi-functional Kdo-transferase encoded by the gene gseA. gseA of Chlamydia psittaci 6BC was cloned and expressed in a rough mutant (Re chemotype) of Escherichia coli (strain F515) that contains an LPS with only two α2→4-linked Kdo residues. Recombinant strains were able to add the immunodominant Kdo residue in a α2→8-linkage to the parental LPS, as determined by SDS–PAGE and Western blot analysis using a monoclonal antibody against the genus-specific epitope. The DNA sequence of gseA was determined and aligned to that published recently for C. trachomatis serovar L2. Most surprisingly, the two deduced amino acid sequences shared only an overall homology of 67%. Thus, gseA exhibits species specificity at the DNA level, whereas its gene product results in the synthesis of a carbohydrate antigen with genus specificity.     

Isolation and structural characterization of an R-form lipopolysaccharide from Yersinia enterocolitica serotype O:8.

The lipopolysaccharide (LPS) of strain 8081-c-R2, a spontaneous R-mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of D-GlcN, D-Glc, L-glycero-D-manno- and D-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1-3) were isolated using high-performance anion-exchange chromatography, the structures of which were determined by compositional analysis and one- and two-dimensional NMR spectroscopy as [carbohydrate structure see text] in which all sugars are pyranoses, and R and R' represent beta-D-Glc (in 1 and 2) and beta-D-GlcN (in 1 only), respectively. D-alpha-D-Hep is D-glycero-alpha-D-manno-heptose, L-alpha-D-Hep is L-glycero-alpha-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI-TOF MS. Its beta-D-GlcN4P-(1-->6)-alpha-D-GlcN-1-->P backbone is mainly tetra-acylated with two amide- and one ester-linked (at O3 of the reducing GlcN) (R)-3-hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3-OH group of the amide-linked (R)-3-hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri- and hexa-acylated lipid A species occur.     

Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J-5 lipopolysaccharide.

The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Escherichia coli J-5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial infections with gram-negative pathogens. The successive de-O-acylation and de-N-acylation of J-5 LPS yielded phosphorylated oligosaccharides which represent the complete carbohydrate backbone. Five compounds were separated by high-performance anion-exchange chromatography and analysed by one-dimensional and two-dimensional homonuclear and heteronuclear 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The main product was a nonasaccharide of the structure alpha-D-Glcp-(1-->3)-[alpha-D-GlcpN- (1-->7)-alpha-L,D-Hepp-(1-->7)]-alpha-L,D-Hepp-(1-->3)-alpha -L, D-Hepp-4P-(1-->5)-[alpha-Kdop-(2-->4)]-alpha-Kdop-(2-- >6)-beta-D-GlcpN-4p- (1-->6)-alpha-D-GlcN-1P wherein all sugars are present as D-pyranoses. Hep and Kdo represent L-glycero-D-manno-heptose and 3-deoxy-D-manno-oct-2-ulosonic acid, respectively. In addition, two octasaccharides and two heptasaccharides were isolated that were partial structures of the nonasaccharide. In one octasaccharide the terminal alpha-D-GlcpN was missing and an additional phosphate group linked to O4 of the branched heptose was present, whereas in the other octasaccharide the side-chain Kdo was missing. In both heptasaccharides the side-chain alpha-D-GlcpN-(1-->7)-L-alpha-D-Hepp-disaccharide was absent; they differed in their phosphate substitution. Whereas both heptasaccharides contained two phosphates in the lipid-A backbone (beta-1,6-linked GlcpN-disaccharide at the reducing end) and one phosphate group at O4 of the first heptose, only one of them was additionally substituted with phosphate at O4 of the second heptose.     

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from a clinical isolate of Yersinia enterocolitica O:9.

Yersinia enterocolitica O:9 strain Ruokola/71-c-PhiR1-37-R possesses mainly rough-type lipopolysaccaride (LPS) and smaller amounts of S-form LPS. Structural analysis of the former is reported here. After deacylation of the LPS, the phosphorylated carbohydrate backbone of the inner core-lipid A region could be isolated by using high-performance anion-exchange chromatography. Its structure was determined by means of compositional and methylation analyses and 1H-, 13C-, and 31P-NMR spectroscopy as: [see text] in which L-alpha-D-Hep is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hep is D-glycero-alpha-D-manno-heptopyranose, and Kdo is 3-deoxy-D-manno-oct-2-ulopyranosonic acid. All hexoses are pyranoses.     

Enzymatic synthesis of β-D-Gal-(1→3)-[β-D-GlcNAc-(1→6)]-α-D-GalNAc-OC6H4NO2-p as a carbohydrate unit of mucin-type 2 core

We have established a synthetic method for obtaining β-D-Gal-(1→3)-[β-D-GlcNAc-(1→6)]-α-D-GalNAc-OC6H4NO2 -p (1), which is a carbohydrate unit of mucin-type 2 core. A β-N-acetyl-D-hexosaminidase from Nocardia orientalis catalyzed the synthesis of the desired compound 1 with its isomers β-D-GalNAc-(1→6)-β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p (2) β-D-GlcNAc-(1→3)-β-D-Glc-(1→3)-α-D-GalNAc-OC6H4NO2-p (3) through N-acetylglucosaminyl transfer from N,N′-diacetylchitobiose and β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p. The enzyme formed the trisaccharides 1, 2, and 3 in 14% overall yield based on β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p as an acceptor substrate, and in the ratio of 44:32:24. In this way, N-acetylglucosaminyl transfer favored O-6 of the acceptor rather than O-6′, and occurred to a lesser extent at O-3′. This reaction was efficient enough to allow a one-pot preparation of the desired carbohydrate unit of mucin-type 2 core. When β-D-Gal-(1→3)-β-D-GalNAc-OC6H4NO2-p was used as an acceptor, the enzyme also synthesized three kinds of trisaccharides in the same regioselectivity with respect to O-6 and O-6′ versus O-3′ of the acceptor.     

Structural elucidation of a novel core oligosaccharide backbone of the lipopolysaccharide from the new bacterial species Agrobacterium larrymoorei

Agrobacterium larrymoorei is a Gram-negative phytopathogenic bacterium, which produces tumours on Ficus benjamina plants and differs from other Agrobacteria both genetically and biochemically. The lipopolysaccharide (LPS) plays an important role in the pathogenesis of Agrobacteria. The present paper is the first report on the molecular primary structure of the core region of an Agrobacterium LPS. The following structure of the core and lipid A carbohydrate backbone of an R-form LPS of A. larrymoorei was determined by chemical degradations and 1D and 2D NMR spectroscopy: [carbohydrate structure: see text] All sugars are alpha-D-pyranoses if not stated otherwise, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Qui3NAcyl is 3,6-dideoxy-3-(3-hydroxy-2,3-dimethyl-5-oxoprolylamino)glucose, GlcAN and GalAN are amides of GlcA and GalA.     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.     

Structural investigations of the galactan of the snail Lymnaea stagnalis

A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d-galactose and 0.9% of nitrogen, but neither l-galactose nor phosphate groups. The [α]_D²⁰ values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra-O-methyl- and 2,4-di-O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri-O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-d-Gal-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-[β-d-Gal-(1→6)]-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, and β-d-Gal-(1→3)-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.     

Structure of the d-galacto-d-mannan isolated from the seeds of Melilotus indica All

A d-galacto-d-mannan ([α]_D +72.0 and d-galactose-to-d-mannose ratio 1:1.14) was isolated from the seeds of Melilotus indica All., syn. M. parviflora Desf. The ¹H- and ¹³C-n.m.r., and i.r. spectra indicated the presence of α-d-galactopyranosyl and β-d-mannopyranosyl residues. Methylation of the polysaccharide, followed by hydrolysis, afforded, 2,3,4,6-tetra-, 2,3,6-tri-, 2,3-di-, and 3,4-di-O-methyl-d-mannose, and 2,3,4,6-tetra- and 2,3,6-tri-O-methyl-d-galactose in the molar ratios of 1:2:22:6:27:3. Periodate oxidation of the polysaccharide, followed by reduction and hydrolysis, gave erythritol (1 mol) and glycerol (1.24 mol). Partial acid hydrolysis of the polysaccharide afforded O-β-d-mannopyranosyl-(1→2)-d-mannopyranose, O-β-d-mannopyranosyl-(1→4)-d-mannopyranose, O-α-d-galactopyranosyl-(1→6)-d-mannopyranose, O-α-d-galactopyranosyl-(1→4)-d-galactopyranose, and O-α-d-galactopyranosyl-(1→6)-O-β-d-mannopyranosyl-(1→4)-d-mannopyranose. A highly branched structure having a mannan backbone composed of 36% of (1→4)- and 10% of (1→2)-linked β-d-mannopyranosyl units is proposed for the galactomannan.     

Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen

Abstract An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia -specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occuring in patient sera. These mAbs recognized Chlamydia -specific epitopes [ α 2→8-linked disaccharide of 3-deoxy- d - manno -octulosonic acid (Kdo) or the trisaccharide α Kdo-(2→8)-→Kdo] or those shared between chlamydial and Re-type LPS ( α Kdo, α →4-linked Kdo disacccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.     

A novel 3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase that removes the outer Kdo sugar of Helicobacter pylori lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Étude complémentaire de la structure de trois galactoxyloglucanes (amyloïdes) de graines

D-Galacto-D-xylo-D-glucans (amyloids) from Balsamina, Tropaeolum, and Tamarindus seeds behave in a similar manner in the presence of various glycosidase preparations: slow depolymerization by enzymes from several germinated or non-germinated seeds, and hydrolysis into monosaccharides and oligosaccharides by commercial cellulase and hemicellulase preparations from fungi. A purified cellulase from Penicillium notatum gave a dialyzable fraction almost exclusively composed of α-D-xylopyranosyl-(1→6)-D-glucose residues and a nondialyzable fraction composed of chains of β-D-(1→4)[withsome (1→3)]-glucopyranosyl residues; β-D-galacto-pyranosyl-(1→2)-α-D-xylosyl groups are linked to some of the β-D-glucosyl residues at 0-6. The presence of (1→3)-linkages in the D-glucan chain of the Balsamina was verified by methylation and sequential periodate oxidation-borohydride reduction; the distribution of the substituents on the D-glucan chain is not regular. The main D-glucan backbone, where the β-D-glucosyl residues are partly linked at 0-6 to β-D-galactosyl-(1→2)-D-xylosyl groups, is linked to D-glucan chains where almost all the D-glucose units are linked at 0-6 by one α-D-xylosyl group. The presence of 3,6-di-O-methyl-D-glucose after permethylation and hydrolysis suggests that the xyloglucan chains are linked to 0-2 of the D-glucosyl units of the galactoxyloglucan backbone.     

Chemical and biological properties of a highly branched β-glucan from edible mushroom Pleurotus sajor-caju

Hot aqueous extraction of the basidiocarps of the mushroom Pleurotus sajor-caju provided a cold water-soluble, gel-like glucan, which was characterized chemically, and its effects on RAW 264.7 cell line (mouse leukaemic monocyte macrophage) activation were determined. NMR spectroscopy, HPSEC, methylation analysis, and a controlled Smith degradation showed it to have a branched structure with a (1→3)-linked β-Glcp main-chain, substituted at O-6 by single-unit β-Glcp side-chains, on the average of two to every third residues of the backbone, with a molar mass of 9.75×10(5)gmol(-1). In macrophage cell culture, the β-glucan induced production of NO and the cytokines TNF-α, IL-1β, these effects being very similar as those of Escherichia coli serotype 0111:B4 Sigma-Aldrich lipopolysaccharide (LPS), although not modifying the response of LPS-activated macrophages. The results suggest that the (1→3), (1→6)-linked β-glucan from P. sajor-caju may have potential for immunological activities, although additional experiments are necessary for a better understanding of the mechanisms involved.     

Lipopolysaccharide biosynthesis without the lipids: recognition promiscuity of Escherichia coli heptosyltransferase I

Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k(cat)/K(m) of 2.7 × 10(6) M(-1) s(-1), compared to a value of 2.2 × 10(5) M(-1) s(-1) for the Kdo(2)-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.     

Constitution of sargassan,a sulphated heteropolysaccharide from sargassum linifolium

The behaviour towards periodate of the brown-algal polysaccharide sargassan before and after partial hydrolysis, alkali treatment, and methanolysis has been studied. Evidence is thereby provided that the sargassan backbone is composed of (1→4)-linked β-D-glucuronic acid and β-D-mannose residues. Heteropolymeric, partially sulphated branches are attached to the backbone, and these branches comprise various proportions of(l→4)-linked, β-D-galactose, β-D-galactose 6-sulphate, and β-D-galactose 3,6-disulphate residues, (1→2)-linked α-L-fucose 4-sulphate residues, and (1→3)-linked β-D-xylose residues.     

Comparative studies of asparagine-linked oligosaccharide structures of rat liver microsomal and lysosomal beta-glucuronidases

The sugar chains of microsomal and lysosomal β-glucuronidases of rat liver were studied by endo-β-N-acetylglucosaminidase H digestion and by hydrazinolysis. Only a part of the oligosaccharides released from microsomal β-glucuronidase was an acidic component. The acidic component was not hydrolyzed by sialidase and by calf intestinal and Escherichia coli alkaline phosphatases, but was converted to a neutral component by phosphatase digestion after mild acid treatment indicating the presence of a phosphodiester group. The neutral oligosaccharide portion of microsomal enzyme was a mixture of five high mannose-type sugar chains: (Manα1 → 2)_0~4 [Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc]. In contrast, lysosomal enzyme contains only Manα1 → 6 (Manα1 → 3) Manα1 → 6(Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc. The result indicates that removal of α1 → 2-linked mannosyl residues from (Manα1 → 2)₄[Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn] starts already in the endoplasmic reticulum of rat liver.