[alpha]1,2-Mannosidases are essential for hybrid and complex N-glycan biosynthesis in mammalian cells (Kornfeld and Kornfeld, 1985; Moremen et al., 1994; Herscovics, 1999a,b). Following the removal of the glucose residues from the Glc3Man9GlcNAc2 precursor structure attached to nascent glycoproteins, ER and Golgi [alpha]1,2-mannosidases catalyze the trimming of the four [alpha]1,2-linked mannose residues. The subsequent action of GlcNAc transferase I initiates complex chain formation and yields the substrate for Golgi [alpha]-mannosidase II which trims the terminal [alpha]1,3- and [alpha]1,6-mannose residues. In some tissues a distinct [alpha]-mannosidase trims Man5GlcNAc2 to Man3GlcNAc2 prior to the action of GlcNAc transferase I (Tulsiani and Touster, 1985; Bonay and Hughes, 1991; Chui et al., 1997). Thereafter, the N-glycan structure is further elaborated by Golgi glycosyltransferases.
[alpha]-Mannosidases have been classified into two groups based on amino acid sequence homology and on biochemical properties (Daniel et al., 1994; Moremen et al., 1994). Class I [alpha]-mannosidases specifically hydrolyze [alpha]1,2-linked mannose residues, and do not cleave substrates such as p-nitrophenyl-[alpha]-d-mannopyranoside. They require calcium for activity and are inhibited by 1-deoxymannojirimycin and kifunensine, but not by swainsonine. In contrast, Class II [alpha]-mannosidases can cleave [alpha]1,2-, [alpha]1,3- and [alpha]1,6-linked mannose residues as well as p-nitrophenyl-[alpha]-d-mannopyranoside and are inhibited by swainsonine, but not by 1-deoxymannojirimycin.
Although several mammalian [alpha]1,2-mannosidases that can remove up to four [alpha]1,2-mannose residues have been purified and cloned (Tabas and Kornfeld, 1979; Tulsiani et al., 1982; Schweden et al., 1986; Tulsiani and Touster, 1988; Forsee et al., 1989; Schweden and Bause, 1989; Bause et al., 1993; Herscovics et al., 1994; Lal et al., 1994; Tremblay et al., 1998), there is significant biochemical evidence for the existence of highly specific mammalian enzymes that trim Man9GlcNAc2 to Man8GlcNAc2 isomer B, the form lacking the middle-arm terminal [alpha]1,2-mannose, but mammalian enzymes with this specificity have not yet been purified or cloned. A mammalian ER [alpha]1,2-mannosidase that forms Man8GlcNAc2 isomer B and is not sensitive to 1-deoxymannojirimycin was described in intact UT-1 cells and in rat hepatocytes (Bischoff et al., 1986) whereas distinct 1-deoxymannojirimycin-sensitive [alpha]1,2-mannosidase activity that processes Man9GlcNAc2 to Man8GlcNAc2 isomer B was observed in the ER of intact COS cells (Rizzolo and Kornfeld, 1988) and in ER and in Golgi rat liver membrane preparations (Tulsiani and Touster, 1988; Weng and Spiro, 1993; Lal et al., 1998). Up to now the yeast ER processing [alpha]1,2-mannosidase is the only enzyme purified (Jelinek-Kelly and Herscovics, 1988; Ziegler and Trimble, 1991) and cloned (Camirand et al., 1991) that specifically trims Man9GlcNAc2 to Man8GlcNAc2 isomer B.
The present work reports the isolation, expression, and properties of a novel human cDNA encoding a Class I [alpha]1,2-mannosidase that specifically converts Man9GlcNAc to Man8GlcNAc isomer B.
Isolation and characterization of a novel human [alpha]1,2-mannosidase cDNA
A novel human [alpha]1,2-mannosidase was identified by searching the EST database with the yeast [alpha]1,2-mannosidase amino acid sequence (Camirand et al., 1991). The consensus sequence of the identified overlapping clones encodes the terminal 50 amino acids of the catalytic domain including two of the most highly conserved Class I [alpha]-mannosidase sequence motifs followed by 589 bp of 3[prime] UTR terminating in a poly A tail. Clones encoding the complete ORF (2.1 kb) and 5[prime] UTR were then isolated by nested 5[prime] RACE from human placenta, liver, and testis cDNAs, as described in Materials and methods. In addition an independent 2.7 kb clone containing the ORF flanked by 50 bp of 5[prime] UTR and 589 bp of 3[prime] UTR terminating with a polyA tail was isolated from a human fetal brain cDNA library. The sequence of the cDNAs obtained from the different sources were identical.
The 2.7 kb cDNA is predicted to encode a 79.5 kDa type II membrane protein with an 85 amino acid cytoplasmic tail, followed by a putative transmembrane domain of about 17 residues, a 'stem" region of about 137 amino acids not required for enzyme activity and a large C-terminal catalytic domain (amino acids 240-699) (Figure
Figure 1. Nucleotide and deduced amino acid sequence of the human [alpha]1,2-mannosidase cDNA. The 5[prime] UTR and ORF sequence were obtained from human placenta, testis, and liver cDNA clones amplified by RACE, and a fetal brain cDNA library clone. The 3[prime] UTR sequence is the consensus of an alignment of EST clones. Bold numbers refer to the deduced amino acid sequence and the numbers in normal type refer to the nucleotide sequence. The conserved Class 1 [alpha]-mannosidase sequence motifs are underlined, and the highly conserved invariant acidic amino acid residues as well as the conserved cysteines common to all class I [alpha]-mannosidases are boxed in gray. The putative transmembrane domain sequence is indicated in bold and underlined. Arrows indicate the starting amino acid residues of the different forms of the recombinant enzyme expressed in Pichia pastoris.
Northern blot analysis indicates that all tissues examined expressed variable levels of a 3 kb transcript (Figure
Figure 2. Northern blot analysis of human [alpha]1,2-mannosidase expression. Random labeled [alpha]1,2-mannosidase EST clone R16652 was hybridized to Northern blots containing 2 µg of poly (A+) RNA from human tissue. The blots were exposed to film to x-ray film for 5 days. Expression of recombinant [alpha]1,2-mannosidase in Pichia pastoris
The [alpha]1,2-mannosidase was expressed as a secreted protein in P.pastoris in order to characterize its enzymatic activity. The catalytic domain starting at either amino acid 169 or 240 was cloned in frame downstream from the [alpha]-factor of the P.pastoris expression vector pPICZ[alpha] A. Following methanol induction of yeast transformed with expression constructs pZ[alpha]ASHM169, pZ[alpha]ASHM240, or pZ[alpha]ASHM240T similar levels of [alpha]1,2-mannosidase activity were detected in the medium, and absent in yeast transformed with the pPICZ[alpha] A vector. Recombinant [alpha]1,2-mannosidase of the expected size (55 kDa) was observed by Western blot analysis at 2-3 days postinduction and decreased thereafter (Figure
Figure 3. Expression of the recombinant [alpha]1,2-mannosidase in P.pastoris. Ten microliters of medium was subjected to reducing SDS-PAGE (10%) and the recombinant Myc tagged [alpha]1,2-mannosidase was visualized by Western blot analysis. Lanes 1-4 correspond to GS115 transformed with pZ[alpha]ASHM240T at 2, 3, 4, and 5 days postinduction, respectively. Lane 5 corresponds to GS115 transformed with vector at 5 days postinduction. Molecular mass standards are indicated on the right. Properties of the human [alpha]1,2-mannosidase
The catalytic properties of the recombinant [alpha]1,2-mannosidase were studied using [3H]Man9GlcNAc as substrate. The enzyme is active over a pH range of 6.3-7.2 with an optimum between 6.5-6.9. Inclusion of at least 0.1 mM Ca2+ in the assay is required to obtain maximum enzyme activity. The enzyme is inhibited 50% by 1 µM EDTA and 100% by 25 µM EDTA. This inhibition is completely reversed by the addition of 2 mM Ca2+ but, not by 2 mM Mn2+, Mg2+, Zn2+, or Co2+. The Km of the recombinant enzyme is 0.4 mM which is similar that of the recombinant yeast [alpha]1,2-mannosidase (0.3 mM) (Lipari and Herscovics, 1994).
The human [alpha]1,2-mannosidase is inhibited by the Class I [alpha]-mannosidase inhibitors, 1-deoxymannojirimycin (IC50 = 75 µM) and kifunensine (IC50 = 70 nM), but is insensitive to the Class II [alpha]-mannosidase inhibitor swainsonine (Table I).
Table I. Specificity of the human [alpha]1,2-mannosidase
The recombinant [alpha]1,2-mannosidase was incubated with [3H]Man9GlcNAc for different periods of time and HPLC analysis showed that the only products formed are Man8GlcNAc and mannose in a time dependent manner (Figure
Figure 4. Time course human [alpha]1,2-mannosidase activity. [3H]Man9GlcNAc was incubated with medium obtained from P.pastoris transformed with pZ[alpha]ASHM169 2 days postinduction. Oligosaccharide product formation was monitored by HPLC as described in Materials and methods and is expressed as a percentage of the total radioactivity recovered.
Figure 5. [1H]-NMR spectrum identifying the Man8GlcNAc isomer produced by the human [alpha]1,2-mannosidase. Spectrum at 600 MHz and 30°C of the anomeric region of the Man8GlcNAc produced by the hydrolysis of Man9GlcNAc by the specific human [alpha]1,2-mannosidase. The resonance corresponding to the anomeric proton of each mannose residue is numbered. Integral values for each of the anomeric protons, except GlcNAc, were obtained. The split resonance signal at 5.104 and 5.075 p.p.m. for residue 7 is characteristic of Man8GlcNAc isomer B. Materials
Oligonucleotides were synthesized by BioCorp (Montréal, Canada). Man9GlcNAc was prepared from soya bean agglutinin and [3H]mannose-labeled Man9GlcNAc from rat liver as described previously (Jelinek-Kelly et al., 1985; Bhattacharyya et al., 1988). Man8GlcNAc isomer B substrate was prepared by digestion of Man9GlcNAc with the yeast [alpha]1,2-mannosidase (Lipari and Herscovics, 1994). 1-deoxymannojirimycin, kifunensine and swainsonine were obtained from Toronto Research Chemicals, Inc. (Downsview, Canada). All other chemicals were reagent grade. Isolation of a novel human [alpha]1,2-mannosidase cDNA
The NCBI dbEST database was searched with the yeast [alpha]1,2-mannosidase amino acid sequence (Camirand et al., 1991) using the tBLASTn algorithm to identify novel [alpha]1,2-mannosidases. Retrieved ESTs were aligned using the DNASTAR SeqMan program (Madison, WI) and EST clones AA631254, R16652, and H46222 encoding the 3[prime] region of the ORF were obtained from Genome Systems Inc. and sequenced. The following nested primers were designed based on the consensus sequence, 5[prime]-CCACAGACCCAGCAAGGTGCC-3[prime] and 5[prime]-CTAGGCAGGGGTCCAGATAGG-3[prime], and used to amplify clones containing additional 5[prime] sequence from human placenta Marathon Ready cDNA (CLONTECH) according to the recommended protocol. A 5 µl aliquot the PCR reactions was subcloned into pCRII using the original TA cloning kit (Invitrogen) and clones encoding [alpha]1,2-mannosidase were identified by hybridization of colony lifts with gene specific 32P-labeled oligonucleotide probes. The longest clones (2.1 kb) were sequenced.
Thereafter, the Gibco 5[prime] RACE System (Version 2) was used to isolate additional 5[prime] sequence. First strand cDNA was synthesized from 2.5 µg of placenta, testis, and liver total RNA (CLONTECH) at 55°C using ThermoScript RT (Gibco) and a gene specific primer (GSP1 5[prime]-GGGCACTTCTGCTCTTCTTGAAG-3[prime]) located within the 5[prime] region of the placenta cDNA clones. Nested 5[prime] RACE amplicons were then generated using gene specific primers (GSP2 5[prime]-ATGACTGTCCTCTGCGGATCTC-3[prime], and GSP3.1 5[prime]-TGTCTTCTGTGACGAAATCTC-3[prime] or GSP3.2 5[prime]-CAGAGCTTTCCAATGGTCAGC-3[prime] or GSP3.3 5[prime]-TCATAGCTCTCGCCAAAGCTCAGC-3[prime]) and Platinum Taq (Gibco) according to the recommended protocol. The amplicons were subcloned into pCR2.1 (Invitrogen), identified by hybridization of colony lifts with gene specific 32P-labeled oligonucleotide probes, and sequenced. In addition, Genome Systems isolated three fetal brain [alpha]1,2-mannosidase cDNA clones (2.7 kb) by a PCR and hybridization cDNA library screen using primers located within the 5[prime] ORF (5[prime]-ATCGGGACTTCACCTCGGTG-3[prime] and 5[prime]-CAGAGCTTTCCAATGGTCAGC-3[prime]). Northern blot analysis
The human [alpha]1,2-mannosidase EST R16652 was labeled with [[alpha]-32P]dCTP (3000 Ci/mmol) using the multiprime DNA labeling kit (Amersham). The probe was hybridized to human multiple tissue Northern blots (CLONTECH) according to the recommended protocol and exposed to x-ray film for 5 days (Kodak). Expression of the catalytic domain in Pichia pastoris
The DNA sequence encoding the soluble catalytic domain of the [alpha]1,2-mannosidase was amplified from a cDNA clone by PCR using the sense oligonucleotide 5[prime]-AAAGAATTCCAGATTAGACCCCCAAGCCAAG-3[prime] containing an EcoRI site and the antisense primer SHMSTOP 5[prime]-AAATCTAGACTAGGCAGGGGTCCAGATAGG-3[prime] containing the stop codon followed by an XbaI site. Expression vector pZ[alpha]ASHM169 was constructed by ligation of the amplicon into the EcoRI/XbaI sites of pPICZ[alpha] A (Invitrogen) in frame with the [alpha]-factor secretion signal. Similarly, shorter untagged (pZ[alpha]ASHM240) and tagged (pZ[alpha]ASHM240T) expression constructs were prepared using sense primer 5[prime]-AAAGAATTCCAGGGCACACCAGTGCATCTG-3[prime] and antisense primers SHMSTOP, and SHMR 5[prime]AAATCTAGAGCAGGGGTCCAGATAGGCAG-3[prime], respectively. Primer SHMR lacks a termination codon thus the C-terminal of the recombinant enzyme was fused to the (His)6 and Myc tags encoded by pPICZ[alpha] A.
Pichia pastoris strain GS115 (his4) (Invitrogen) was transformed by electroporation with 10 µg of the expression constructs linearized with PmeI. Transformants were grown and assayed for [alpha]1,2-mannosidase activity as described previously (Herscovics and Jelinek-Kelly, 1987; Tremblay et al., 1998). [alpha]-Mannosidase assays
To characterize the recombinant [alpha]1,2-mannosidase, medium containing the enzyme was concentrated 7.5-fold using centrifugal filters (Millipore) and equilibrated in 40 mM PIPES pH 6.5. Two microliters of the concentrated medium was incubated with [3H]mannose-labeled Man9GlcNAc at 37°C and the amount of released [3H]mannose was determined by the Con A/PEG precipitation method (Herscovics and Jelinek-Kelly, 1987).
Divalent cation requirements were studied by including 0.05 mM EDTA in duplicate assays in the absence or presence of 2 mM CaCl2, ZnCl2, MnCl2, MgCl2, and CoCl2. The enzyme was incubated for 2 h with 5000 c.p.m. of [3H]Man9GlcNAc in 40 mM PIPES pH 6.5, 1 mg/ml BSA, and 1 mM NaN3.
The Km was determined by Lineweaver-Burk analysis. Duplicate 30 min assays contained Man9GlcNAc substrate (0.05-0.5 mM), 5000 c.p.m. of [3H]Man9GlcNAc, 1 mM CaCl2, 40 mM PIPES pH 6.5, 1 mg/ml BSA, and 1 mM NaN3.
The effects of the Class I [alpha]-mannosidase inhibitors 1-deoxymannojirimycin and kifunensine, and the Class II [alpha]-mannosidase inhibitor swainsonine were investigated by preincubating the enzyme on ice for 30 min with the inhibitor in 40 mM PIPES pH 6.5, 1 mM CaCl2, 1 mg/ml BSA, and 1 mM NaN3. Substrate (0.8 mM Man9GlcNAc and 20,000 c.p.m. of [3H]Man9GlcNAc) was then added to the duplicate assays and the mixtures were incubated at 37°C for 1 h.
The time dependent formation of products was analyzed by assaying the recombinant enzyme at 37°C in a 30 µl mixture containing 3.4 mM Man9GlcNAc, 30,000 c.p.m. [3H]Man9GlcNAc, 44 mM potassium phosphate pH 6.5, 1 mg/ml BSA, 1 mM NaN3 and 12 µl of unconcentrated medium. At 0, 1, 2, 4, 8, and 19.5 h one-sixth of the reaction mixture was collected. The products were resolved by HPLC, and identified by comparing their elution to that of the [14C]Glc3Man9GlcNAc internal standard, as described previously (Romero et al., 1985). High Resolution [1H]-NMR analysis
Medium containing the human [alpha]1,2-mannosidase was incubated at 37°C with 600 µg Man9GlcNAc and 105 c.p.m. [3H]Man9GlcNAc in 44 mM potassium phosphate buffer pH 6.5 containing 1 mg/ml BSA and 1 mM NaN3. The incubation was supplemented with additional enzyme after 8 and 22.5 h, and terminated after 28.5 h by boiling for 3 min. An aliquot of the sample was analyzed by HPLC to show that most of the sample was transformed to Man8GlcNAc. The sample was then chromatographed on a Bio-Gel P-6 column (1 × 109 cm) equilibrated in deionized water. Fractions containing the oligosaccharide product were pooled, lyophilized, resuspended in D2O and lyophilized four times, and stored over P2O5 in a vacuum desiccator. The [1H]-NMR spectra were recorded at Université de Montréal NMR Facility in 5 mm tubes using a 600 MHz Bruker spectrometer at 30 and 70°C with the acetone chemical shift set to 2.225 p.p.m. with respect to 4,4-dimethyl-4-silapentane sulfonate. Polyacrylamide gel electrophoresis Western blotting
SDS-PAGE was performed using the Bio-Rad Mini-Protean II apparatus as described by Laemmli (Laemmli, 1970). Western blots were prepared by transferring proteins onto a nitrocellulose membrane (Schleicher and Schuell), and expression of the Myc-tagged recombinant [alpha]1,2-mannosidase was detected using the monoclonal Anti-myc Antibody (Invitrogen) and visualized by the ECL Western blotting detection system (Amersham). DNA sequencing and alignments
Manual sequencing was performed using the Pharmacia T7 and Deaza sequencing kits. The Sheldon Biotechnology Centre Automated Sequencing Facility (McGill University, Montréal, Canada) employed the ABI prism dye terminator and thermo sequenase fluorescent labeled primer cycle sequencing kits and the samples were run on the ABI 373A (Perkin Elmer) and ALFexpress (Amersham Pharmacia Biotech) sequencers respectively. The DNASTAR SeqMan program (Madison, WI) was used to assemble the sequences into contigs. The deduced amino acid sequences were aligned using the BestFit and Publish programs (ver. 9.1) from the University of Wisconsin Genetics Computer Group (Madison, WI).
We thank Dr. Nancy L.Shaper and Dr. Pedro Romero for valuable advice and Mr. Barry Sleno for technical assistance. This work was supported by an operating grant from the Medical Research Council of Canada. L.O.T. was a recipient of a scholarship for graduate studies from the Medical Research Council of Canada.
ER, endoplasmic reticulum; RT, reverse transcriptase; ORF, open reading frame; RACE, rapid amplification of cDNA ends; GSP, gene-specific primer; YPD, yeast peptone dextrose; BMGY, buffered glycerol-complex; BMMY, buffered methanol complex; HPLC, high-performance liquid chromatography.
The cDNA encoding the human specific [alpha]1,2-mannosidase has been independently isolated and characterized (Gonzalez et al., 1999) and shown to be localized in the endoplasmic reticulum of transfected mammalian cells. However, the ORF of the cDNA reported in the latter manuscript is lacking 36 amino acids at the N-terminal compared to the sequence reported in the present paper (AF148509).
Inhibitor
Activitya (% of control)
1-Deoxymannojirimycin
10 µM
84
50 µM
62
500 µM
10
Kifunensine
0.05 µM
67
0.10 µM
28
0.50 µM
0
Swainsonine
5 µM
100
10 µM
100
100 µM
100
Materials and methods
Acknowledgments
Abbreviations
Note added in proof
References
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