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J. Biol. Chem., Vol. 275, Issue 41, 31655-31660, October 13, 2000

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Characterization of a cDNA Encoding a Novel Human Golgi 1,2-Mannosidase (IC) Involved in N-Glycan Biosynthesis^*

Linda O. Tremblay and Annette Herscovics^§

From the McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada

Received for publication, June 7, 2000, and in revised form, July 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I 1,2-mannosidases was isolated. The enzymatic properties of the novel 1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. 1,2-Mannosidase IC sequentially hydrolyzes the 1,2-linked mannose residues of [³H]mannose-labeled Man₉GlcNAc to form [³H]Man₆GlcNAc and a small amount of [³H]Man₅GlcNAc. The enzyme requires calcium for activity and is inhibited by both 1-deoxymannojirimycin and kifunensine. The order of mannose removal was determined by separating oligosaccharide isomers formed from pyridylaminated Man₉GlcNAc₂ by high performance liquid chromatography. The terminal 1,2-linked mannose residue from the middle branch is the last mannose removed by the enzyme. This residue is the mannose cleaved from Man₉GlcNAc₂ by the endoplasmic reticulum 1,2-mannosidase I to form Man₈GlcNAc₂ isomer B. The order of mannose hydrolysis from either pyridylaminated Man₉GlcNAc₂ or Man₈GlcNAc₂ isomer B differs from that previously reported for mammalian Golgi 1,2-mannosidases IA and IB. The full-length 1,2-mannosidase IC was localized to the Golgi of MDBK and MDCK cells by indirect immunofluorescence. Northern blot analysis showed tissue-specific expression of a major transcript of 3.8 kilobase pairs. The expression pattern is different from that of human Golgi 1,2-mannosidases IA and IB. Therefore, the human genome contains at least three differentially regulated Golgi 1,2-mannosidase genes encoding enzymes with similar, but not identical specificities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1,2-Mannosidases play an essential role in the maturation of N-glycans to hybrid and complex structures in mammalian cells (for reviews, see Refs. 1-3). They remove the four 1,2-linked mannose residues from Man₉GlcNAc₂, following cleavage of glucose from Glc₃Man₉GlcNAc₂. Thus, 1,2-mannosidases provide the Man₅GlcNAc₂ substrate required for GlcNAc transferase I that initiates formation of complex and hybrid N-glycans. They belong to class I -mannosidases (family 47 of the glycosyl hydrolase classification (Ref. 4)) that have been conserved through eukaryotic evolution. The 1,2-mannosidases are type II transmembrane proteins with amino acid similarity throughout their large C-terminal catalytic domains. They are inverting calcium-dependent glycosyl hydrolases that are inhibited by 1-deoxymannojirimycin and kifunensine. However, they have different N-terminal regions and intracellular localizations. A class I 1,2-mannosidase localized to the ER¹ of mammalian cells has been cloned (5, 6). It has the same properties as the yeast ER 1,2-mannosidase, the structure of which has recently been determined by x-ray crystallography (7). The ER 1,2-mannosidase removes a single specific mannose residue from Man₉GlcNAc₂ to form Man₈GlcNAc₂ isomer B that lacks the terminal 1,2-mannose from the middle branch of the oligosaccharide. Two class I Golgi 1,2-mannosidases, IA and IB, that are about 65% identical in amino acid sequence have also been cloned from mammalian cells (8-11). These Golgi enzymes remove the four 1,2-linked mannose residues from Man₉GlcNAc₂ to yield Man₅GlcNAc₂. Their specificity is complementary to that of the ER 1,2-mannosidase since the mannose residue cleaved by the ER enzyme is the last residue removed by the two Golgi 1,2-mannosidases (12). The major difference between Golgi 1,2-mannosidase IA and IB is their tissue- and cell-specific expression as shown by Northern blot analysis of human and murine tissues (9-11), and by immunolocalization in cells of the rat testis (13). In addition, there is some difference in their specificity with Man₉GlcNAc as substrate (12).

In the present work, the characterization of Golgi human 1,2-mannosidase IC, a novel member of the mammalian class I 1,2-mannosidases is reported. This enzyme displays a distinct pattern of tissue-specific expression and trims Man₉GlcNAc₂ to Man₅GlcNAc₂, forming different high mannose oligosaccharide intermediates from those previously observed for mammalian Golgi 1,2-mannosidases IA and IB.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Oligonucleotides were synthesized by BioCorp (Montréal, Canada). The C-terminal peptide was synthesized and conjugated to keyhole limpet hemocyanin by the Sheldon Biotechnology Centre (McGill University, Montréal, Canada). [³H]Mannose-labeled Man₉GlcNAc was prepared from rat liver and Man₉GlcNAc from soybean agglutinin as described previously (14, 15). Man_(9-6)GlcNAc₂-PA oligosaccharides were purchased from Takara Shuzo Co. (Otsu, Japan). Kifunensine, 1-deoxymannojirimycin, and swainsonine were obtained from Toronto Research Chemicals, Inc. (Downsview, Canada).

Isolation of Human 1,2-Mannosidase IC cDNA-- ESTs encoding 1,2-mannosidase IC were identified by querying the NCBI dbEST data base with the yeast 1,2-mannosidase amino acid sequence (16) using the tBLASTn algorithm. Most of the 1,2-mannosidase IC ESTs identified were catalogued in UniGene file Hs.8910. The ESTs encoding the 3' end of the transcript were aligned, and clones T54452, AA437353, N30588, H67812, H68084, and W19722, spanning the length of the consensus sequence (1.3 kb), were obtained from Genome Systems Inc. and sequenced. Primers within the 5' region of the consensus sequence (5'-ACCTGAACGTGAGCGGAGAAG-3'; 5'-CCTGGTTGCCAGA GAGTTCAG-3') were used to screen a fetal brain cDNA library by PCR and hybridization (Genome Systems). The isolated clone (2.2 kb) contained 0.9 kb of additional 5' sequence. In addition, a partially sequenced 2.9-kb EST clone (AA017666) was identified when the NCBI Human UniGene file was reorganized and listed clone sizes. The clone was obtained from Research Genetics and entirely sequenced.

Northern Blot Analysis-- Human 1,2-mannosidase IC EST clone W19722 was labeled with [-³²P]dATP (3000 Ci/mmol) using the multiprime DNA labeling kit (Amersham Pharmacia Biotech). Human multiple tissue Northern blots (CLONTECH) were hybridized with the 1,2-mannosidase IC probe according to the recommended protocol and exposed to x-ray film (Eastman Kodak Co.).

1,2-Mannosidase IC Antibodies-- Rabbits were immunized with 0.5 mg of the keyhole limpet hemocyanin-conjugated synthetic C-terminal peptide NHSDSSGRAWGRH emulsified in Freund's complete adjuvant and boosted 5 weeks later with the same peptide in Freund's incomplete adjuvant. Serum was collected 12 days later. Antipeptide antibodies were affinity-purified (17) using a column prepared by coupling the peptide to cyanogen bromide-activated Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia Biotech).

Expression of the Catalytic Domain in Pichia pastoris-- The DNA sequence encoding the catalytic domain (amino acids 165-630) was amplified by PCR using a sense primer containing a KpnI site (5'-AAAGGTACCCAGGAGCCCCAGAGCCAAGTG-3') and an antisense primer with a XbaI site following the stop codon (5'-AAATCTAGATCAGTGTCTGCCCCAGGCTCTG-3'). The amplicon was inserted into the KpnI/XbaI sites of pPICZ A (Invitrogen) in frame with the -factor signal sequence yielding the expression construct pZAHMIC493. The expression construct (10 µg) was linearized with PmeI and electroporated into P. pastoris strain GS115 (his4) (Invitrogen), and transformants were grown as described previously (11). Clones expressing recombinant 1,2-mannosidase were identified by assays with [³H]Man₉GlcNAc (18).

SDS-PAGE and Western Blotting-- Medium containing recombinant 1,2-mannosidase, with and without Endo H (New England Biolabs) treatment, was subjected to SDS-PAGE (19) using the Bio-Rad Mini-Protean II apparatus. The proteins were then transferred onto a nitrocellulose membrane (Schleicher & Schuell). Recombinant 1,2-mannosidase was detected using affinity-purified peptide antibodies visualized by the ECL detection system (Amersham Pharmacia Biotech).

1,2-Mannosidase Assays-- Medium containing the recombinant 1,2-mannosidase was concentrated 10-fold using centrifugal filters (Millipore) and equilibrated in 100 mM PIPES, pH 6.8. The 1,2-mannosidase IC activity was assayed by incubating 2 µl of the concentrated medium with 5000-20,000 cpm of [³H]mannose-labeled Man₉GlcNAc in 50 mM MES, pH 5.9, 1 mg/ml BSA, 10 mM CaCl₂, and 1 mM NaN₃ at 37 °C. The amount of released [³H]mannose was assessed by the concanavalin A/polyethylene glycol precipitation method (18).

HPLC Analysis of Oligosaccharide Products-- To characterize the products formed from Man₉GlcNAc, 14 µl of medium containing recombinant 1,2-mannosidase (10-fold concentrated) was incubated at 37 °C with 35,000 cpm [³H]Man₉GlcNAc and 3.3 mM Man₉GlcNAc in a total volume of 35 µl of 50 mM MES, pH 5.9, containing 1 mg/ml BSA, 10 mM CaCl₂, and 1 mM NaN₃. The assay mixture was supplemented with fresh enzyme at 8 and 24 h. Samples (1/7) were analyzed at 0, 1, 2, 4, 8, 24, and 48 by HPLC on an Aminospherisorb column (Waters Corp.), as described previously (20).

To characterize the oligosaccharide isomers, 10 µl of medium was incubated with either 50 pmol of either Man₉GlcNAc₂-PA or Man₈GlcNAc₂-PA in 50 mM MES, pH 5.9, containing 1 mg/ml BSA, 10 mM CaCl₂, and 1 mM NaN_3. The assay mixtures were supplemented with fresh enzyme at 24 h. Samples (1/6) were collected at 0, 2, 4, 8, 24, and 48 h. The products were first fractionated according to size by HPLC on a TSK-Gel Amide 80 column (4.6 × 250 mm, TosoHaas), eluted isocratically at 1 ml/min with a 1:1 v/v mix of acetonitrile, water, 500 mM acetic acid, pH 7.3 (75:15:10, v/v/v) and acetonitrile, water, 500 mM acetic acid, pH 7.3 (50:40:10, v/v/v). The pH of the 500 mM acetic acid was adjusted to 7.3 with triethylamine. Oligosaccharides were monitored with a Varian model 360 fluorescent detector at an excitation of 310 nm and an emission of 380 nm. The oligosaccharide products were collected manually and lyophilized. The isomers present in each fraction were then resolved by HPLC on a MicroPak-SP C18 column (4.6 × 150 mm, Varian), eluted isocratically at 1 ml/min with 100 mM acetic acid containing 0.025% n-butyl alcohol adjusted to pH 4 with triethylamine. The isomers were monitored at an excitation of 320 nm and an emission of 400 nm. The identity of the products was determined by comparing their elution to that of standard Man_9-5GlcNAc₂-PA.

Localization of 1,2-Mannosidase IC in MDBK and MDCK Cells by Immunofluorescence-- The ORF sequence was amplified by PCR using a sense primer containing a HindIII site and Kozak sequence (5'-AAAAAAGCTTCCACCATGCTCATGAGGAAAGTG-3'). The antisense primer containing a NotI site was either 5'-AAAAAAAAGCGGCCGCGAGTGTCTGCCCCAGGCTCTG-3' or 5'-AAAAAAAAGCGGCCGCTCAGTGTCTGCCCCAGGCTCTG-3' (including a stop codon). The ORF amplicons were cloned into the HindIII/NotI sites of pMH (Roche Molecular Biochemicals) in frame with the C-terminal hemagglutinin tag, yielding the tagged (pMHHMICT) and untagged (pMHHMICS) constructs.

MDBK and MDCK cells were grown to 70% confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 5 µg/ml gentamicin. Following trypsinization, 1.4 × 10⁶ cells in PBS containing 20 mM HEPES were transiently transfected by electroporation with 10 µg of either pMHHMICT or pMHHMICS. The cells were grown overnight on coverslips in 35-mm dishes, and the following morning the medium was changed. At 24 and 48 h after electroporation, the cells were washed twice with PBS, fixed with 3% paraformaldehyde (prewarmed to 37 °C) for 10 min, washed twice with PBS, permeabilized for 2 min with 0.2% Triton X-100 in PBS, and blocked with fetal calf serum for 1 h at room temperature. Following one wash with PBS containing 0.2% Tween 20 (PBST), the cells were incubated for 2 h with the primary antibodies diluted in PBST containing 3% BSA. The primary antibodies were mouse monoclonal anti-hemagglutinin antibody HA11 (BAbCo) (1:1000 or 1:2000), affinity-purified rabbit polyclonal anti-1,2-mannosidase IC antibodies (1:50, 1:100, or 1:200), or affinity-purified rabbit polyclonal anti-bovine 1,4-galactosyltransferase antibodies (1:200) (gift of Dr. Joel Shaper, John Hopkins, Baltimore, MD (Ref. 21)). Following five washes with PBST, the primary antibodies were detected by incubation for 1 h with affinity-purified CY2 conjugated anti-mouse IgG (1:400) or goat anti-rabbit IgG conjugated with rhodamine (tetramethylrhodamine B isothiocyanate) (1:800) (Jackson ImmunoResearch). The cells were then washed five times with PBST and mounted onto slides in Immuno-Fluore mounting medium (ICN). They were viewed with a Nikon Eclipse 800 epifluorescence microscope and photographed with TMax P3200 film (Kodak).

DNA Sequencing and Alignments-- DNA sequencing was done by the Sheldon Biotechnology Centre (McGill University, Montréal, Canada) using the ABI prism dye terminator sequencing kit and ABI 373A sequencer, or with the Thermo Sequenase fluorescent labeled primer cycle sequencing kit (Perkin Elmer) and ALFexpress sequencer. Sequencing was also done by Bio S&T Inc. (Montréal, Canada) using the SequiTherm EXCEL II kit (Epicenter Technologies) and a Long Readir 4200 sequencer. Sequences were assembled into contigs with the DNASTAR SeqMan program (Madison, WI), and deduced amino acid sequences were aligned using the PileUp and Gap programs (version 10.0) from the University of Wisconsin Genetics Computer Group (Madison, WI).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Characterization of Human 1,2-Mannosidase IC-- Human ESTs encoding 1,2-mannosidase IC were identified by querying the EST Data Base as described under "Experimental Procedures." The 1.3-kb consensus sequence obtained upon aligning the ESTs encoded the C-terminal region of the catalytic domain (216 amino acids) including two of the highly conserved class I 1,2-mannosidase amino acid sequence motifs, 991 base pairs of flanking 3'-untranslated region and a poly(A) tail. Thereafter, a cDNA clone encoding the entire catalytic domain (1.1 kb) as well as the 3'-untranslated region (1 kb) was identified by a PCR screen of a fetal brain cDNA library using primers within the 5' region of the consensus sequence. In addition, a partially sequenced EST clone identified in the UniGene data base was completely sequenced (2.9 kb) and shown to encode the entire ORF.

The 1,2-mannosidase IC cDNA (2.9 kb) is predicted to encode a 70.9-kDa type II membrane protein with a short cytoplasmic tail of about 22 amino acid residues, a transmembrane domain of 22 residues and a large C-terminal domain (Fig. 1). The C-terminal domain contains a proline-rich "stem" region (amino acids 45-164) not required for enzyme activity, followed by the catalytic domain (amino acids 165-630). The latter encodes class I 1,2-mannosidase signature motifs (see the Carbohydrate-Active Enzymes server, available via the world wide web) and the nine invariant acidic amino acids and cysteine residues shown to be essential for the activity of the yeast class I 1,2-mannosidase (23, 24). Three potential N-glycosylation sites are located within the catalytic domain. 1,2-Mannosidase IC is about 54% identical to the human (X74837, AF027156), murine (U04299, U03458), and porcine (Y12503) 1,2-mannosidases IA and IB, and 38% identical to the human ER 1,2-mannosidase (AF145732, AF148509) amino acid sequences (5, 6, 8-11, 25).

View larger version (86K): [in this window] [in a new window]   Fig. 1.   Nucleotide and deduced amino acid sequence of human 1,2-mannosidase IC. Numbers at the right in normal font refer to the nucleotide sequence and those in bold to the deduced amino acid sequence. The conserved class I 1,2-mannosidase motifs are indicated in bold and underlined, and invariant acidic amino acid residues and cysteines are circled. The putative transmembrane domain is denoted in bold and underlined by a dotted line. The starting amino acid residue of the recombinant enzyme expressed in P. pastoris is indicated by a diamond, and asterisk marks potential N-glycan sites.

1,2-Mannosidase Expression in Human Tissues-- Northern blot analysis revealed variable expression of a major 3.8-kb transcript in most tissues with the exception of lung, muscle, and pancreas (Fig. 2). Remarkably high levels of the major transcript are expressed in the placenta. The ovary, liver, and placenta also expressed minor transcripts of about 2.4 kb, and several tissues expressed low levels of a 5.7-kb transcript. Both the transcript sizes and expression differ from those reported for human 1,2-mannosidase IA (3.8, 4.3 kb) and IB (7.5, 9.5 kb) (11) with the exception of the 5.7-kb transcript, but this same size transcript is different since it is expressed differentially in the tissues analyzed.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Human 1,2-mannosidase IC expression. [-32P]dATP-labeled 1,2-mannosidase EST clone W19722 was hybridized to Northern blots containing 2 µg of poly(A+) RNA isolated from human tissues. The blots were exposed to x-ray film for 5 days. Molecular size markers are indicated beside each blot.

Expression of Recombinant 1,2-Mannosidase IC in P. pastoris-- The catalytic domain starting at amino acid 165 was cloned in the P. pastoris expression vector pPICZ A in frame with the -factor signal sequence. 1,2-Mannosidase activity was detected in the medium 2 days following induction with methanol of yeast cells transformed with the resulting construct pZAHMIC493. No activity was found in the medium of cells transformed with the empty vector pPICZ A. The secreted recombinant 1,2-mannosidase consists of a 55-kDa and a heterogeneous 67-kDa form. Treatment with Endo H gives rise to a single band of the expected size of 52 kDa (Fig. 3). These results indicate that one glycoform only acquires core N-glycans, whereas the other contains outer chains with an average of about 16 residues per core structure, assuming all three sites are equally glycosylated.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Recombinant 1,2-mannosidase IC expressed in P. pastoris. Ten microliters of medium (concentrated 10-fold), treated with or without Endo H, was subjected to 10% SDS-PAGE (reducing) and visualized by Western blotting. Lanes 1 and 2, GS115 transformed with pZAHMIC493; lane 3, GS115 transformed with pPICZ A, at 48 h after induction. Molecular mass markers are indicated on the right.

Properties of Recombinant 1,2-Mannosidase IC-- The enzymatic properties of recombinant 1,2-mannosidase IC were analyzed using [³H]mannose-labeled Man₉GlcNAc as substrate. The enzyme has a pH optimum of about 5.9 and requires the addition of calcium for maximum activity. Inhibition of the enzyme by preincubating with 50 µM EDTA is reversed by the addition of 10 mM Ca²⁺, but not by 10 mM Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, or Fe²⁺. The 1,2-mannosidase IC activity is inhibited by the class I -mannosidase inhibitors 1-deoxymannojirimycin (IC₅₀ = 250 µM) and kifunensine (IC₅₀ = 0.5 µM), but not by the class II -mannosidase inhibitor swainsonine (Table I). Therefore, 1,2-mannosidase IC has all the properties ascribed to class I 1,2-mannosidases.

View this table: [in this window] [in a new window]   Table I Effects of inhibitors on human 1,2-mannosidase IC activity

Specificity of Human 1,2-Mannosidase IC-- The enzyme was incubated with [³H]mannose-labeled Man₉GlcNAc, and the products obtained at different times were resolved by HPLC. The recombinant enzyme catalyzed the stepwise removal of mannose from Man₉GlcNAc to form Man₆GlcNAc and a small amount of Man₅GlcNAc (Fig. 4). Man₉GlcNAc incubated for 48 h with medium of yeast transformed with the vector alone was not hydrolyzed.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Time course of 1,2-mannosidase IC hydrolysis of Man9GlcNAc. [3H]Man9GlcNAc was incubated with medium (10-fold concentrated) from P. pastoris transformed with pZAHMIC493 at 2 days after induction. The products, Man9GlcNAc (), Man8GlcNAc (), Man7GlcNAc (), Man6GlcNAc (), and Man5GlcNAc (), were resolved by HPLC on an Aminospherisorb column as described under "Experimental Procedures." The results are expressed as a percentage of the total radioactivity recovered at each time point.

To determine the order of mannose removal, the enzyme was incubated with Man₉GlcNAc₂-PA. The Man_8-6GlcNAc₂ intermediates were first fractionated according to size by HPLC (data not shown). Each oligosaccharide fraction was then further fractionated into its component isomers by HPLC on a reverse phase column. Hydrolysis of Man₉GlcNAc₂ yields primarily a single Man₈GlcNAc₂ isomer (about 90%), equivalent amounts of two Man₇GlcNAc₂ isomers, and a single Man₆GlcNAc₂ isomer that were identified by comparison with elution of standard oligosaccharides-PA (Fig. 5, A-C). These results indicate that the terminal 1,2-linked mannose residue on the middle arm is the last to be removed. Since Man₈GlcNAc₂ isomer B is formed by human ER 1,2-mannosidase I, Man₈GlcNAc₂-PA isomer B was also incubated with 1,2-mannosidase IC. In this case the enzyme first cleaves the terminal mannose on the 1,3-branch of the substrate, yielding essentially a single Man₇GlcNAc₂ isomer (about 85%). Thereafter, equivalent amounts of two Man₆GlcNAc₂ isomers (Fig. 5, D and E) were formed by the hydrolysis of either of the two remaining 1,2-linked mannose residues. Thus, the order of mannose removal from Man₈GlcNAc₂-PA was identical to the order observed for the Man₉GlcNAc₂-PA.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Oligosaccharide intermediates formed by 1,2-mannosidase IC. The Man8-6GlcNAc2-PA isomers formed from Man9GlcNAc2-PA (A-C) and Man8GlcNAc2-PA (D and E) were resolved by HPLC on a C18 column as described under "Experimental Procedures." The structures of the substrates are shown above the profiles. Arrows indicate the elution position of standards whose structures are shown above the arrows. , 1,2-linked mannose residues; , 1,3- and 1,6-linked mannose residues; , GlcNAc2-PA.

Immunolocalization of 1,2-Mannosidase IC in Transfected MDBK and MDCK Cells-- The full-length 1,2-mannosidase IC was expressed in MDBK and MDCK cells to determine its subcellular localization by indirect immunofluorescence. Punctate perinuclear Golgi staining was detected in cells 24-48 h after transfection with both the hemagglutinin tagged (pMHHMICT) and native (pMHHMICS) 1,2-mannosidase IC (Fig. 6). The staining pattern shows that 1,2-mannosidase IC is in the Golgi since it co-localizes with endogenous Golgi 1,4-galactosyltransferase. No immunofluorescence was observed with pre-immune serum, secondary antibodies alone, or cells transfected with the pMH vector.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Localization of 1,2-mannosidase IC in MDBK cells. MDBK cells were transiently transfected with either hemagglutinin tagged (A-D) or native (E and F) 1,2-mannosidase IC. The cells were fixed and stained with monoclonal HA11 hemagglutinin antibody (A and C), and polyclonal 1,4-galactosyltransferase antibodies (B) or polyclonal 1,2-mannosidase IC antibodies (D and E). The HA11 (A and C) and polyclonal (B, D, and E) antibodies were detected with CY2- and rhodamine-conjugated antibodies, respectively. Phase contrast of the cell in panel E is presented in panel F.

Genomic Organization and Chromosomal Localization-- The 1,2-mannosidase IC gene contains 12 exons encoded by GenBank clones AL031280 and AL020996. These clones overlap by 2.3 kb within the intronic region between exons 2 and 3. The gene is localized on chromosome 1p35.1-36.13 and spans 167 kb of genomic sequence between the markers D1S2843 and D1S417 on Gene Map 98 (26). The intron and exon boundaries of the coding region are identical to those found in the human 1,2-mannosidase IA gene, which spans 188 kb on chromosome 6q22 (UniGene Hs.2750). The reported genomic organization of the 1,2-mannosidase IB (11) localized on human chromosome 1p13 differs from 1,2-mannosidase IA and IC at a few positions within the ORF (Fig. 7), particularly at the N terminus, which is encoded by two exons.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Organization of human Golgi 1,2-mannosidase genes. 1,2-Mannosidase IA is encoded by GenBank genomic clones AL078600 (exons 1-3) and AL022722 (exons 4-12). The intron and exon boundaries within the coding region are identical to those of the 1,2-mannosidase IC gene (AL031280 (exons 1 and 2) and AL020996 (exons 3-12)). The 1,2-mannosidase IB gene organization (11) differs at the indicated positions. Coding region exons are indicated by numbered boxes, introns are denoted by dotted lines, and solid lines represent the 5'- and 3'-untranslated regions. Numbers above the boxes correspond to the position of the 3' nucleotide in the exons relative to the first nucleotide of the ORF.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present results demonstrate the existence of a previously unsuspected third mammalian Golgi 1,2-mannosidase derived from a distinct gene. This enzyme is capable of trimming high mannose oligosaccharides to Man₅GlcNAc₂ during N-glycan biosynthesis. Human 1,2-mannosidase IC requires calcium for activity and is inhibited by 1-deoxymannojirimycin and kifunensine; thus, it possesses the characteristic properties of class I 1,2-mannosidases. The amino acid sequence of the catalytic domain is similar to previously described mammalian Golgi 1,2-mannosidases IA and IB, but the cytoplasmic tail and stem region sequence differ. Furthermore, 1,2-mannosidase IC displays a distinct tissue-specific expression pattern and order of 1,2-linked mannose removal (Fig. 8).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Comparison of Man9GlcNAc2 and Man8GlcNAc2 isomer B trimming by mammalian Golgi 1,2-mannosidases. Products formed by Golgi 1,2-mannosidase IC reported here are compared with those previously reported for Golgi 1,2-mannosidases IA and IB (12) and ER 1,2-mannosidase I (5, 6). Enzymes highlighted in black above the arrows represent the major pathway(s), whereas those beneath the arrow indicate a minor pathway (<30% of product). , 1,2-linked mannose residues; , 1,3-and 1,6-linked mannose residues; , GlcNAc2.

Human Golgi 1,2-mannosidases IA, IB, and IC are encoded by independent genes on chromosomes 6q22, 1p13, and 1p35-36, respectively. Gene duplication occurring late in evolution probably gave rise to the mammalian Golgi 1,2-mannosidase gene family (27) since the positions of the intron and exon boundaries within the gene are very similar. However, in both humans and mice these genes are independently regulated, thus giving rise to distinct patterns of expression (9-11, 13).

1,2-Mannosidase IC readily hydrolyzes three of the four 1,2-linked mannose residues of Man₉GlcNAc₂ and slowly cleaves the remaining terminal 1,2-linked mannose residue on the middle branch (Fig. 8, upper section). The enzyme produces the same Man₈GlcNAc₂ isomer as 1,2-mannosidase IA (12) and then forms equivalent amounts of two Man₇GlcNAc₂ isomers. One of the Man₇GlcNAc₂ isomers is also formed by recombinant murine 1,2-mannosidases IA and IB (12), and purified rat Golgi 1,2-mannosidase (28), whereas the other isomer (not produced by IA or IB) is formed by recombinant insect (29) and fungal (30) 1,2-mannosidases, and is an inferred intermediate of purified porcine 1,2-mannosidase (31). The human Golgi 1,2-mannosidase activities are complementary to the human ER 1,2-mannosidase (5, 6) since they hydrolyze the terminal 1,2-linked mannose of the middle arm of Man₉GlcNAc₂ last. Hydrolysis of Man₈GlcNAc₂ isomer B by 1,2-mannosidase IC proceeds readily to Man₅GlcNAc₂ (Fig. 8, lower section). The mannose residues are removed in the same order observed for Man₉GlcNAc₂. However, this order differs from that observed with murine 1,2-mannosidases IA and IB (12). These results demonstrate that 1,2-mannosidase IC has a unique specificity that differs from that of mammalian Golgi 1,2-mannosidases IA and IB.

Recent x-ray crystallographic studies of the yeast ER 1,2-mannosidase indicate the active site of class I 1,2-mannosidases is located within an ()₇ barrel with many non-conserved amino acids interacting with different parts of the oligosaccharide substrate (7). Furthermore, mutation of one of these amino acids was demonstrated to change the specificity of the yeast ER 1,2-mannosidase (22). Therefore, it is likely that the variations in the order of mannose removal by the various class I 1,2-mannosidases is largely determined by the differences in non-conserved amino acids interacting with the oligosaccharide substrate within the barrel. The relative expression of mammalian Golgi 1,2-mannosidases with slightly different specificities can thus provide different high mannose oligosaccharide isomers with possible variation in recognition functions.

	ACKNOWLEDGEMENTS

We thank Dr. Nancy Shaper and Dr. Pedro Romero for advice, Dr. Joel Shaper for the 1,4-galactosyltransferase antibody, Barry Sleno for technical assistance, and Michel Massaad and Dr. Burkhard Becker for assistance with immunofluorescence microscopy.

	FOOTNOTES

^* This work was supported by an operating grant from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF261655.

Recipient of a scholarship for graduate studies from the Medical Research Council of Canada.

^§ To whom correspondence should be addressed: McGill Cancer Center, 3655 Promenade Sir-William-Osler, Montréal, Québec, Canada H3G 1Y6. Tel.: 514-398-3533; Fax: 514-398-6769; E-mail: annette@ med.mcgill.ca.

Published, JBC Papers in Press, July 27, 2000, DOI 10.1074/jbc.M004935200

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; BSA, bovine serum albumin; ORF, open reading frame; HPLC, high performance liquid chromatography; PA, pyridylamino; PAGE, polyacrylamide gel electrophoresis; Endo H, endo--N-acetylglucosaminidase H; PIPES, 1,4-piperazinediethanesulfonic acid; contig, group of overlapping clones; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline plus Tween 20; kb, kilobase pair(s); EST, expressed sequence tag; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; MDCK, Madin-Darby canine kidney; MDBK, Madin-Darby bovine kidney.

	REFERENCES

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