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Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human [alpha]1,2-mannosidase gene involved in N-glycan maturation
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human [alpha]1,2-mannosidase gene involved in N-glycan maturation
Class I [alpha]1,2-mannosidases play an essential role in the elaboration of complex and hybrid N-glycans in mammalian cells. Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and lymphocyte cDNAs. One of these was related to the previously cloned human and murine [alpha]1,2-mannosidase IA whereas the other was very similar to murine [alpha]1,2-mannosidase IB. Northern blot analysis of human tissues with these two [alpha]1,2-mannosidase probes revealed very different patterns of tissue-specific expression. Similar tissue-specific expression of [alpha]1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues. A human placenta cDNA library was screened and PCR of brain, placenta, and lymphocyte cDNAs was performed in order to isolate the human [alpha]1,2-mannosidase IB cDNA. This cDNA encodes a type II membrane protein of 73 kDa that is 94% identical in amino acid sequence to the murine [alpha]1,2-mannosidase IB (Herscovics et al., 1994, J. Biol. Chem., 269, 9864-9871). A truncated soluble form of the human [alpha]1,2-mannosidase IB lacking its N-terminal transmembrane domain was expressed as a secreted protein in Pichia pastoris. The recombinant enzyme was incubated with [3H]Man9GlcNAc and [3H]Man8GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that [3H]Man9GlcNAc was readily converted to [3H]Man6GlcNAc and much more slowly to [3H]Man5GlcNAc, whereas [3H]Man8GlcNAc was rapidly trimmed to [3H]Man5GlcNAc. The human [alpha]1,2-mannosidase IB gene was isolated from a P1 human genomic library and shown to be at least 60 kb in size and to contain at least 13 exons. The gene was localized by fluorescence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers. These results show that there are at least two Class I [alpha]1,2-mannosidases in the human and murine genomes with very distinct transcriptional regulation in different tissues.
Key words: processing/chromosome 1p13/[alpha]1,2-mannosidase/ N-glycan
Introduction
[alpha]-Mannosidases play an important role at different stages in the maturation of N-glycans in mammalian cells (Kornfeld and Kornfeld, 1985; Moremen et al., 1994; Herscovics, 1998). Following transfer of the Glc3Man9GlcNAc2 precursor to nascent polypeptides and removal of the three glucose residues in the endoplasmic reticulum the concerted action of [alpha]1,2-mannosidases present in the endoplasmic reticulum and Golgi cleave up to four [alpha]1,2-linked mannose residues to form Man5GlcNAc2. This oligosaccharide is a substrate of GlcNAc transferase I, the first glycosyltransferase that initiates the branches of complex N-glycans. Thereafter Golgi [alpha]-mannosidase II can cleave the terminal [alpha]1,3- and [alpha]1,6-mannose residues to yield GlcNAcMan3GlcNAc2. In some tissues, there is another [alpha]-mannosidase activity that can remove the [alpha]1,3- and [alpha]1,6-mannose residues from Man5GlcNAc2 to form Man3GlcNAc2 which is also a substrate of GlcNAc transferase I (Tulsiani and Touster, 1985; Bonay and Hughes, 1991; Chui et al., 1997). Further maturation of the carbohydrate side chains can then proceed through the action of various Golgi glycosyltransferases. Studies with specific [alpha]1,2-mannosidase inhibitors such as 1-deoxymannojirimycin and kifunensine demonstrate that removal of the [alpha]1,2-linked mannose residues from the oligosaccharide precursor during the early stages of the N-glycan processing pathway is required for the formation of complex and hybrid oligosaccharides (Fuhrmann et al., 1985; Romero et al., 1986; Elbein, 1991). However, thenumber of ER and Golgi [alpha]-mannosidases involved in N-glycan maturation in mammalian cells is still unclear (Moremen et al., 1994; Herscovics, 1998).
[alpha]-Mannosidases have been purified and cloned from different sources and based on amino acid sequence similarity, these enzymes have been classified into Class I and Class II [alpha]-mannosidases with distinct sequences and enzymatic properties (Moremen et al., 1994). Class I [alpha]-mannosidases have been conserved throughout eukaryotic evolution, and have been cloned from yeast (Camirand et al., 1991), human (Bause et al., 1993), rabbit, mouse (Herscovics et al., 1994; Lal et al., 1994), insects (Kerscher et al., 1995; Kawar et al., 1997), fungi, and mold (Yoshida et al., 1993; Inoue et al., 1995). Class I [alpha]-mannosidases specifically cleave [alpha]1,2-mannose residues, are inhibited by 1-deoxymannojirimycin but not by swainsonine, and do not use aryl [alpha]-d-mannosides as substrates. On the other hand, Class II [alpha]-mannosidases that include Golgi [alpha]-mannosidase II (Moremen and Robbins, 1991) and lysosomal [alpha]-mannosidase (Nebes and Schmidt, 1994; Liao et al., 1996), are not as specific and cleave [alpha]1,2-, [alpha]1,3- and [alpha]1,6-linked mannose residues. Class II [alpha]-mannosidases are inhibited by swainsonine but not by 1-deoxymannojirimycin.
Mammalian [alpha]1,2-mannosidases have been purified from rat (Tabas and Kornfeld, 1979; Tulsiani et al., 1982; Tulsiani and Touster, 1988), rabbit (Forsee et al., 1989), calf (Schweden et al., 1986), and pig (Schweden and Bause, 1989) livers. The rat liver Golgi [alpha]1,2-mannosidase could be separated into two forms, IA and IB, by ion exchange chromatography and gel filtration (Tabas and Kornfeld, 1979; Tulsiani et al., 1982), but it was not established whether these are isoforms derived from the same gene or whether they arise from different genes since the rat liver Golgi enzymes have not been cloned. A mammalian [alpha]1,2-mannosidase was first cloned using amino acid sequence of the purified pig liver enzyme to design oligonucleotides for RT-PCR and the resulting cDNA probe was used to screen a human kidney cDNA library (Bause et al., 1993). The human cDNA thus isolated encodes a type II membrane protein of 71 kDa capable of cleaving [alpha]1,2-linked mannose residues. The protein localizes to the Golgi following transfection of COS cells (Bieberich and Bause, 1995). More recently, a pig liver [alpha]1,2-mannosidase cDNA was also cloned (Bieberich et al., 1997). It encodes a 73 kDa type II membrane protein whose sequence is very similar to that of the human kidney enzyme, but it has an N-terminal region different from that of the human enzyme and localizes to the endoplasmic reticulum in pig hepatocytes (Roth et al., 1990) and in transfected COS cells (Bieberich et al., 1997). Similarly, using primers derived fromamino acid sequence obtained from the purified rabbit liver enzyme, RT-PCR was used to isolate the murine [alpha]1,2-mannosidase IA cDNA encoding a 73 kDa type II membrane protein that is about90%identical in amino acid sequence to the cloned human kidney enzyme (Lal et al., 1994). At the same time, a distinct murine cDNA termed [alpha]1,2-mannosidase IB was also cloned using amino acid sequences that are conserved in all Class I [alpha]1,2-mannosidases to design degenerate primers for PCR (Herscovics et al., 1994). Murine [alpha]1,2-mannosidase IA and IB cDNAs encode proteins that are 65% identical in amino acid sequence. Both proteins localize to the Golgi following transfection of mammalian cells in culture. Southern blot analysis of mouse genomic DNA indicates that they are derived from different genes (Herscovics et al., 1994). The gene encoding murine [alpha]1,2-mannosidase IB was recently characterized and localized to mouse chromosome 3F2 while another related gene or pseudogene was localized to mouse chromosome 4A13 (Campbell Dyke et al., 1997).
Since our previous studies (Herscovics et al., 1994; Campbell Dyke et al., 1997) indicated the presence of an [alpha]1,2-mannosidase gene family in the mouse genome, the present work was undertaken to determine whether several members of this family also exist in the human genome. We describe the isolation and characterization of a novel human cDNA and its corresponding gene, encoding a protein very similar in sequence and properties to the murine [alpha]1,2-mannosidase IB. We also demonstrate that there are at least two human [alpha]1,2-mannosidases encoded by genes on different chromosomes that have very different patterns of expression in different human tissues.
Results
Cloning and characterization of human [alpha]1,2-mannosidase IB cDNA
All the class I [alpha]1,2-mannosidases cloned to date contain several highly conserved amino acid sequence motifs in the catalytic domain that can be used to clone [alpha]1,2-mannosidases from different species. cDNA fragments representing human [alpha]1,2-mannosidases were amplified by PCR from placenta and lymphocyte cDNAs using degenerate primers based on two of these highly conserved amino acid sequences. Products of the expected size (about 660 bp) were isolated from each tissue and subcloned into the TA vector. Sequencing of random clones revealed two distinct amplicon sequences, PCRIA and PCRIB. The deduced amino acid sequence of PCRIA is very similar to regions of the previously isolated human kidney [alpha]1,2-mannosidase cDNA (Bause et al., 1993) and murine [alpha]1,2-mannosidase IA (Lal et al., 1994), and only about 70%identical to the corresponding region of murine [alpha]1,2-mannosidase IB (Herscovics et al., 1994). On the other hand, the deduced amino acid sequence of PCRIB is >90% identical to the corresponding region of murine [alpha]1,2-mannosidase IB and only 70% identical to either the human or murine [alpha]1,2-mannosidase IA. Thus, the PCRIB cDNA is derived from a novel human [alpha]1,2-mannosidase whereas PCRIA most likely corresponds to the previously reported human kidney [alpha]1,2-mannosidase (Bause et al., 1993). It should be noted that the human (Bause et al., 1993) as well as the pig and calf [alpha]1,2-mannosidases (Bause et al., 1992; Bieberich and Bause, 1995; Bieberich et al., 1997) have been called Man9-mannosidases. However, from their sequence similarities they belong to the [alpha]1,2-mannosidase IA subclass.
Using PCRIB as a probe, a [lambda]gt11 placenta cDNA library was screened in order to isolate the full length human [alpha]1,2-mannosidase IB cDNA. A large number of clones were isolated, and only those large enough to contain significant portions of the ORF outside of the probe region were characterized. Eight independent clones ranging in size from 1.4-3.5 kb were sequenced. As none contained a complete ORF, the library was rescreened with a cDNA fragment derived from the most 5[prime] region obtained. The seven largest clones (1.3-1.6 kb)were sequenced but none of the placenta cDNA clones overlapped sufficiently to obtain a complete ORF. Furthermore these partial cDNAs were heterogenous and some appeared to be the product of alternate splicing while others contained incompletely excised introns.
The sequence of the 5[prime] and 3[prime] UTR of the partial placenta cDNA clones were used to design primers to amplify the entire ORF by PCR. The cDNA sequence shown in Figure
Figure 1. Nucleotide and deduced amino acid sequence of human [alpha]1,2-mannosidase IB cDNA. The 5[prime] and 3[prime] UTR were obtained from [lambda] cDNA clones, and the sequence of the ORF was obtained from brain, placenta and lymphocyte PCR amplicons. The numbers in normal type on the right refer to the nucleotides with position 1 corresponding to the initiation codon, and the numbers in bold refer to the deduced amino acid sequence indicated by the single letter code under the nucleotide sequence. Amino acid differences between human and mouse [alpha]1,2-mannosidase IB are indicated beneath the human sequence. The underlined sequence in bold corresponds to the putative transmembrane domain. The conserved regions that were used for the design of degenerate PCR primers are underlined with a dotted line, and the diamond marks the putative N-glycosylation site.
Expression of [alpha]1,2-mannosidases in human and mouse tissues
The expression pattern of the two [alpha]1,2-mannosidases was examined in different human tissues. Hybridization of the PCRIB probe to Northern blots under stringent conditions revealed the tissue specific expression of several transcripts ranging in size from about 3.5-9.5 kb (Figure
Figure 2. Northern blot analysis of the expression of human [alpha]1,2-mannosidase IB and IA.Northern blots containing 2 µg of poly (A+) RNA from human tissues were hybridized with random labeled [alpha]1,2-mannosidase IB cDNA probe PCRIB (A) and [alpha]1,2-mannosidase IA cDNA probe PCRIA (B). The blots were exposed to x-ray film for 5 days.
Quantitation of the relative levels of class I human [alpha]1,2-mannosidase transcripts by densitometric analysis of the autoradiograms indicate that [alpha]1,2-mannosidase IB expression greatly exceeds that of [alpha]1,2-mannosidase IA in some tissues such as placenta and pancreas, whereas [alpha]1,2-mannosidase IA transcripts are expressed at a much higher level than [alpha]1,2-mannosidase IB in other tissues such as liver, spleen, testis, ovary, intestine, and leukocytes. In other tissues, there were similar levels of both sets of transcripts (Figure
Figure 3. Quantitation of [alpha]1,2-mannosidase IA and IB mRNA transcripts in various human and mouse tissues. The transcripts shown in Figure 2 for human, and the mouse [alpha]1,2-mannosidase IA and IB transcripts on the blot shown in Figure 6 of Herscovics et al., 1994 were quantitated as described in Materials and methods. The values are expressed as a ratio of the intensity of human [alpha]1,2-mannosidase transcripts to [beta]-actin transcripts (A) and mouse [alpha]1,2-mannosidase transcripts to glyceraldehyde-3-phosphate dehydrogenase transcripts (B).
Northern blot analysis of mouse tissues also indicates tissue specific expression of mouse [alpha]1,2-mannosidase IA and IB. Several [alpha]1,2-mannosidase IB transcripts ranging in size from 4.2-8.7 kb were observed and in most tissues the major transcripts were 4.2, 5.1, 6.4, and 8.7 kb (Herscovics et al., 1994). Two [alpha]1,2-mannosidase IA transcripts of 2.8 and 5.3 kb were detected in mouse tissues (unpublished observations) and are slightly larger in size than the reported 2.7 and 4.8 kb rat [alpha]1,2-mannosidase IA transcripts (Lal et al., 1994). Of the mouse tissues analyzed, the brain, lung, thymus, and colon displayed a relatively high level of expression of [alpha]1,2-mannosidase IB transcripts, whereas the liver, kidney, and spleen were enriched in [alpha]1,2-mannosidase IA (Figure Figure 4. Expression of human [alpha]1,2-mannosidase IB in P.pastoris. Ten microliters of medium was subjected to 10% SDS-PAGE (reducing) and visualized by Western blotting. Lane 1 corresponds to DIPY115 transformed with pPICZ[alpha] A at 72 h post induction. Lanes 2-5 correspond to DIPY115 transformed with pZHMIB175 at 0, 24, 48, and 72 h post induction, respectively. The positions of the molecular mass standards are indicated on the right.
Enzymatic activity of human [alpha]1,2-mannosidase IB expressed in Pichia pastoris
In order to determine whether the cloned human [alpha]1,2-mannosidase IB is enzymatically active, the cDNA sequence encoding its soluble domain beginning at amino acid 59 was isolated by PCR and cloned in frame with the [alpha]-factor of the P.pastoris expression vector pPICZ[alpha] A to yield pZHMIB175. Recombinant [alpha]1,2-mannosidase IB was expressed as a secreted fusion protein in the methylotrophic yeast P.pastoris.The DIPY115 strain of P.pastoris was used for expression because it has a disruption in the carboxypeptidase Y gene that reduces the proteolytic activity present in the medium(Ohi et al., 1996). [alpha]1,2-Mannosidase activity was detected in the medium following methanol induction of the yeast transformed with pZHMIB175 but not in control cells transformed with pPICZ[alpha] A. Western blot analysis of the proteins secreted into the medium indicates that recombinant [alpha]1,2-mannosidase of the expected size (67 kDa) is present at 24 h and that its level increases at 48 and 72 h post induction of yeast transformed with pZHMIB175, but it is not present in the medium obtained from cells transformed with the control vector pPICZ[alpha] A (Figure
Figure 5. Time course of human [alpha]1,2-mannosidase IB activity. The medium obtained from P.pastoris transformed with pZHMIB175 following 48 h post induction was concentrated 60-fold and 20 µl was incubated with [3H]Man9GlcNAc (A) or [3H]Man8GlcNAc (B) for different times, as indicated. The oligosaccharide products were resolved by HPLC and the amount of each product obtained is expressed as a percentage of the total radioactivity recovered.
The enzymatic activity of the recombinant human [alpha]1,2-mannosidase IB was studied using [3H]mannose-labeled Man9GlcNAc and Man8GlcNAc as substrates, and the reaction products were resolved by HPLC. Incubation of Man9GlcNAc with [alpha]1,2-mannosidase IB resulted in a time-dependent removal of [alpha]1,2-linked mannose residues with Man6GlcNAc as the major product and a small amount of Man5GlcNAc after 8 h of incubation (Figure 
Organization and chromosome localization of human [alpha]1,2-mannosidase IB
In order to characterize the genomic organization of human [alpha]1,2-mannosidase IB a human P1 genomic library was screened by PCR. Three genomic clones encoding the 5[prime] region of the cDNA (clones 8586, 8587, 8588) and a single clone encoding the 3[prime] end of the gene (clone 8543) were isolated. These were characterized by restriction endonuclease digestion, Southern blotting and sequencing. The genomic coding sequences obtained are exact matches of the human [alpha]1,2-mannosidase IB cDNA clones. The intron-exon boundaries were identified by sequencing (Table I) and were found to conform to the GT-AG rule (Breathnach and Chambon, 1981; Shapiro and Senapathy, 1987). The human [alpha]1,2-mannosidase IB gene is comprised of at least 13 exons (Figure
Table I.
Figure 6. Organization of the human [alpha]1,2-mannosidase IB gene and alignment of P1 genomic clones. The exons are represented by numbered white boxes and the position of the introns are indicated by numbered vertical bars. The numbers above the bars correspond to the cDNA nucleotide position of the 5[prime] base of the exon. Arrows indicate the region spanned by each of the isolated P1 genomic clones. The human [alpha]1,2-mannosidase IB gene was mapped using a 30 kb insert obtained from genomic clone 8543 for fluorescence in situ hybridization. Positive hybridization signals at chromosome 1p13 were noted in 20 out of 20 well-spread metaphase cells. Signals were visualized on both homologs in 90% of the positive spreads (Figure
Figure 7. Chromosomal localization of the [alpha]1,2-mannosidase IB gene. Fluorescence in situ hybridization to human metaphase chromosomes using biotinylated inserts of the P1 genomic clone 8543. Human [alpha]1,2-mannosidase IB is localized to chromosome 1p13.
Discussion
In the present work we report the isolation and characterization of the cDNA and gene encoding human [alpha]1,2-mannosidase IB that can trim [alpha]1,2-mannose residues from high mannose oligosaccharides during processing of N-glycans. We also demonstrate that there are at least two distinct human [alpha]1,2-mannosidases arising from different genes and displaying very different patterns of tissue specific expression. The novel human [alpha]1,2-mannosidase IB is encoded by a gene that we have localized by fluorescence in situ hybridization to chromosome 1p13 whereas the human [alpha]1,2-mannosidase IA (also called Man9-mannosidase) cloned by Bause et al. (Bause et al., 1993) resides on chromosome 6 according to the UniGene database (UniGene Hs. 2750 ). Thus the human genome contains at least two functional members of the Class I [alpha]1,2-mannosidase gene family, [alpha]1,2-mannosidase IA and IB, that encode enzymes that are 64%identical in amino acid sequence. The most important difference observed between [alpha]1,2-mannosidase IA and IB is their pattern of expression in different tissues. This differential expression is evident on Northern blots of human tissues and is also observed on Northern blots of mouse tissues. Furthermore, a different pattern of expression of [alpha]1,2-mannosidase IA and IB has also been demonstrated by immunolocalization in different cell types of the rat testis and epididymis. In the testis, differential expression of the two enzymes is dependent on the stage of cellular differentiation whereas in the epididymis it appears to be region-specific (Igdoura et al., 1996). It seems therefore that the expression of these two genes is independently regulated, suggesting that perhaps the two enzymes may have somewhat different functions in N-glycan maturation.
We have shown that a recombinant form of human [alpha]1,2-mannosidase IB readily cleaves three of the four [alpha]1,2-linked mannose residues of Man9GlcNAc to yield Man6GlcNAc and that the fourth mannose is hydrolyzed at a much slower rate to yield Man5GlcNAc. Similar observations were made in specificity studies of the pig liver [alpha]1,2-mannosidase (Schweden and Bause, 1989; Bause et al., 1992) and with the calf liver enzyme only production of Man6GlcNAc2 from Man9GlcNAc2 has been reported (Schweden et al., 1986). The fact that Man8GlcNAc isomer B is much more readily converted to Man5GlcNAc, suggests that the mannose residue that is relatively resistant to hydrolysis by the human [alpha]1,2-mannosidase is the [alpha]1,2-mannose residue specifically hydrolyzed by the Saccharomyces cerevisiae [alpha]1,2-mannosidase (Byrd et al., 1982; Jelinek-Kelly et al., 1985). Similar observations were made with both recombinant murine [alpha]1,2-mannosidase IA and IB, which were shown to more readily convert Man8GlcNAc isomer B than Man9GlcNAc to Man5GlcNAc (Lal et al., 1998). Furthermore, in that study detailed analysis of the products using high resolution 1H-NMR have clearly identified the resistant [alpha]1,2-linked mannose as being the terminal mannose residue of the middle arm. The same residue was also shown by acetolysis to be the last one removed following incubation of labeled Man9GlcNAc with a purified rat liver [alpha]1,2-mannosidase preparation (Tabas and Kornfeld, 1979). The fact that these various [alpha]1,2-mannosidases have a preference for Man8GlcNAc isomer B as substrate may have physiological importance since there is evidence for the existence of ER [alpha]1,2-mannosidase activity in mammalian cells capable of forming isomer B (Bischoff et al., 1986; Weng and Spiro, 1993).
Characterization of the human [alpha]1,2-mannosidase IB gene revealed conservation of genomic organization between the mouse and human genes. Since exon 8 was not found in the genomic clones the possibility of an additional intron being present cannot be ruled out, but all splice junctions were identical in human and mouse genomic sequence. Some evidence was obtained from sequencing of placenta cDNA clones to suggest the existence of isoforms as a result of alternate splicing. Such alternate splicing and heterogeneity of 5[prime] and 3[prime] UTR most likely contribute to the different transcripts observed on Northern blots. Additional work will be required to identify the different [alpha]1,2-mannosidase isoforms and to determine the genetic control of their expression.
The human gene is localized to human chromosome 1p13, a region syntenic to mouse chromosome 3F2 where the murine gene was located (Campbell Dyke et al., 1997). It is of interest that chromosome 1p13 commonly contains aberrations in a variety of human cancers(Sreekantaiah et al., 1993; Johansson et al., 1994; Mathew et al., 1994; Reifenberger et al., 1994; Mitelman et al., 1997),particularly breast cancer (Mitchell and Santibanez-Koref, 1990; Bièche et al., 1993; Nagai et al., 1995), suggesting that alteration of the [alpha]1,2-mannosidase IB gene may possibly contribute to the modifications of N-glycan structures observed in malignant cells.
Materials and methods
Isolation of [alpha]1,2-mannosidase I cDNA probes
[alpha]1,2-Mannosidase probes were generated from human placenta and lymphocyte cDNAs (Kelley W. Moremen, University of Georgia) by PCR using the degenerate oligonucleotide sense primers, 5[prime]-GA(T/C) (T/A)(C/G)I TT(T/C) TA(T/C) GA(A/G) TA(T/C) (T/C)TI (T/C)T(A/G/T/C) AA-3[prime] and antisense primers 5[prime]-(A/G)TG (A/G/T/C)GC (T/C)TC (A/G/T/C)GT (A/G)TT (A/G)AA-3[prime]corresponding to conserved class I [alpha]1,2-mannosidase amino acid sequences DSFYEYLLK and FNTEAH (Herscovics et al., 1994), respectively. The 50 µl reaction mixtures contained 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.001% gelatin, 1 µM of each primer, 400 µM of each dNTP, and 2.5 U of Taq polymerase, and were overlaid with 50 µl of mineral oil. The PCR was performed in a Perkin-Elmer thermal cycler using the following program: 94°C for 3 min, 35 cycles of 1 min at 92°C, 1 min at 50°C, 3 min at 72°C, and a 3 min final extension step at 72°C. The PCR amplicon was subcloned into pCR II using the original TA cloning kit (Invitrogen)and random clones were sequenced. Two amplicons with distinct sequences, PCRIA and PCRIB, were obtained.
Isolation of [alpha]1,2-mannosidase IB cDNA
Partial [alpha]1,2-mannosidase IB cDNA clones were isolated from a human placenta [lambda]gt11 cDNA library (Dr. Morag Park, McGill University) that was screened by hybridization under stringent conditions (Ausubel et al., 1987) first with 32P-labeled PCRIB and then with a 32P-labeled Sal I/Stu I cDNA (nucleotides -87 to 352 in Figure
The entire ORF of [alpha]1,2-mannosidase IB was amplified by two successive rounds of hot start PCR using nested primers derived from the 5[prime] and 3[prime] UTR sequence of the partial placenta cDNA library clones. Human brain, placenta (Clontech), and lymphocyte (Dr. Kelley W. Moremen, University of Georgia) cDNA served as template for the primary amplification in 50 µl mixtures containing 50 mM Tris-HCl pH 9.2, 1.75 mM MgCl2, 16 mM (NH4)2SO4, 0.715 U Taq/Pwo polymerase (Boehringer Mannheim), an AmpliWax gem (Perkin-Elmer), 10 ng of template cDNA,0.2 µM of sense primer, 5[prime]-GAGTTTTCCCATTTTGGCC-3[prime] (nucleotides -102 to -84), and 0.2 µM of antisense primer, 5[prime]-AGGTGAGAATGGTCCTTCTG-3[prime] (nucleotides 1955-1936). Following 30 cycles of 1 min at 94°C, 1 min at 55°C, 3 min at 68°C, and a 10 min final extension at 68°C, 2 µl of the primary reaction served as the template for a second amplification using the nested sense primer, 5[prime]-AGTATTCTCTCCAAGAGCG-3[prime] (nucleotides -25 to -7), and the nested antisense primer, 5[prime]-CTTCTGGAACTGCTTTCATC-3[prime] (nucleotides 1941-1922). The ORF amplicon was subcloned into pCR IIand random clones from each tissue were sequenced.
Northern blot analysis
[alpha]1,2-Mannosidase cDNA probes PCRIA and PCRIB were labeled with [[alpha]-32P]dCTP (3000 Ci/mmol) using the Amersham multiprime DNA labeling kit. The labeled probes were hybridized to human multiple tissue Northern blots under stringent conditions (Clontech) following the recommended protocol and exposed to x-ray film. Thereafter, the filters were stripped and hybridized with a human [beta]-actin cDNA probe labeled with [[alpha]-32P]dCTP (3000 Ci/mmol). Autoradiograms were analyzed by scanning densitometry using Whole Band Analyzer software version 2.4 (Bio Image, Ann Arbor, MI) to quantitate the intensity of the observed autoradiographic bands. The relative levels of the [alpha]1,2-mannosidase transcripts were normalized to the [beta]-actin transcript.
Northern blot analysis of mouse tissues was also performed under stringent conditions. The blot was first hybridized with the ORF of mouse [alpha]1,2-mannosidase IB cDNA as shown in Figure
Expression vector construction
The region of the [alpha]1,2-mannosidase IB cDNA encoding the soluble domain of the enzyme (nucleotides 175-1926) was amplified by hot start PCR using a sense primer, 5[prime]-ATAGCGGCCGCGACTCTTCAAAACACAAACGC-3[prime],containing a NotI site, and an antisense primer, 5[prime]-TATTCTAGATCATCGAACAGCAGGATTACC-3[prime],containing a XbaI site. The 50 µl reaction contained 50 mM Tris-HCl pH 9.2, 1.75 mM MgCl2, 16 mM (NH4)2SO4, 0.715 U Taq/Pwo polymerase, 2 ng of template cDNA, 0.2 µM of each primer, and an AmpliWax gem. Twenty-four cycles of 1 min at 94°C, 1 min at 55°C, 2.5 min at 68°C, and a 3 min final extension step at 68°C were conducted. The PCR amplicon was subcloned into pCR II,sequenced, digested with NotI and SpeI, and ligated into the NotI/XbaI sites of pPICZ[alpha] A (Invitrogen) in frame with the [alpha]-factor secretion signal to produce the vector pZHMIB175 containing the Sh ble zeocin resistance gene. The ligation mixtures were transformed into DH10[beta] and selected on low salt LB medium containing 25 µg/ml of Zeocin.
Expression in Pichia pastoris
P.pastoris strain DIPY115 (Dr. Akio Miyabe, Green Cross Corp., Osaka) (Ohi et al., 1996) derived by disruption of the carboxypeptidase Y gene of the parent strain GS115 (his4) (Cregg et al., 1985) was transformed by electroporation with 10 µg of pZHMIB175 linearized with SacI. The transformants were grown on YPD plates containing 1 M sorbitol and 100 µg/ml of Zeocin, and clones were selected for [alpha]1,2-mannosidase expression using [3H]mannose-labeled Man9GlcNAc as substrate, as described previously (Herscovics and Jelinek-Kelly, 1987). Recombinant P.pastoris clones were grown overnight to an OD600 of 2-10 in BMGY medium buffered with 100 mM potassium phosphate pH 6.5. [alpha]1,2-Mannosidase expression was induced by resuspending the cells to an OD600 of 1 in BMMY medium buffered with 100 mM potassium phosphate pH 6.5 containing 1 M sorbitol, and 0.5% methanol was added every 24 h.
[alpha]-Mannosidase assays
Fresh medium containing the recombinant human [alpha]1,2-mannosidase IB was concentrated up to 60-fold using centrifugal ultrafilters (Millipore), and assayed in a 25 µl reaction mixture containing 25,000 c.p.m. of [3H]mannose-labeled Man9GlcNAc, 3.4 mM Man9GlcNAc, 1 mg/ml BSA, 10 mM potassium phosphate pH 6.8, 1 mM NaN3, and 20 µl of concentrated media. One-fifth of the reaction mixture was analyzed at 0, 1, 2, 4, and 8 h. In addition, Man8GlcNAc isomer B prepared by digestion of Man9GlcNAc with the yeast specific [alpha]1,2-mannosidase (Lipari and Herscovics, 1994) was used as substrate in a parallel incubation. The reaction products were fractionated by HPLC as described previously (Romero et al., 1985) and 1 ml fractions were collected and counted for radioactivity. The 3H-labeled oligosaccharide products were identified by their elution compared to that of the [14C]Glc3Man9GlcNAc internal standard as described previously (Romero et al., 1985).
Isolation of genomic clones
The human P1 phage genomic library was screened by Genome Systems, Inc. by PCR and colony hybridization using two pairs of human [alpha]1,2-mannosidase IB specific primers. The optimal PCR conditions determined using 100 ng of template genomic DNA were 10 mM Tris-HCl pH 9, 50 mM KCl, 3 mM MgCl2, 0.64 mM of each dNTP, 2.5 µM of each primer, and 1.25 U of Taq polymerase. Following an initial 3 min denaturation at 94°C, 30 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and a 10 min final extension step at 72°C were conducted. Clones 8586, 8587, and 8588 encoding the 5[prime] end of the gene were isolated using sense primer H334, 5[prime]-CGAGCTGATCATGAGAAGGC-3[prime] and antisense primer H516, 5[prime]-TGGGTCTCCACCACGTATTC-3[prime]. Clone 8543 encoding the 3[prime] end of the gene was isolated using sense primer H1305, 5[prime]-TAAGAAGTCTCGTGGAGGTC-3[prime] and antisense primer H1490, 5[prime]-GACTCATGACAAGTACGTGC-3[prime] (The primer number corresponds to the base number in Figure
Southern blot analysis of P1 genomic clones
The P1 clones were transduced into the Cre- host NS3516 and plasmid DNA was isolated using the alkaline lysis protocol as recommended by Genome Systems, Inc. The genomic DNA was digested with EcoRI and BamHI, and analyzed by Southern blotting (Ausubel et al., 1987). Restriction endonuclease digestion products encoding exons were identified by hybridization with 32P-labeled oligonucleotide probes, subcloned into pBluescript II KS(-), and fragments containing exons were sequenced to identify intron-exon junctions.
Localization of gene by fluorescence in situ hybridization
Chromosomal mapping was performed at the Mapping Resource Centre of the Canadian Genome Analysis and Technologies Program (Toronto, Ontario) using clone 8543 for fluorescence in situ hybridization (Lichter et al., 1990) to normal human chromosomes counterstained with propidium iodide and DAPI. The probe assignment was determined by the analysis of 20 well spread metaphases, and biotinylated probe was detected with avidin-fluorescein isothiocyanate (FITC). Images of the metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometrics, Tucson, AZ). Separate images of DAPI banded chromosomes (Heng and Tsui, 1993) and of FITC targeted chromosomes were obtained. The acquired hybridization signals were merged using image analysis software and pseudo colored blue (DAPI), and yellow (FITC) (Boyle et al., 1992), and overlaid electronically.
Polyacrylamide gel electrophoresis Western blotting
SDS-PAGE was performed as described by Laemmli (Laemmli, 1970) using the Bio-Rad Mini-Protean II apparatus. For Western blot analysis, proteins were transferred onto a nitrocellulose membrane (Schleicher and Schuell), [alpha]1,2-mannosidase IB was detected using rabbit polyclonal antiserum raised against the mouse C-terminal peptide (amino acid residues 628-641) and visualized by the ECL Western blotting detection system (Amersham).
DNA sequencing
DNA sequencing was done by the dideoxy chain termination method using the Pharmacia T7 and Deaza sequencing kits. The SeqMan program of DNASTAR (Madison, WI) was used to assemble the sequence contigs and the cDNA sequences of the class I [alpha]1,2-mannosidases were aligned using the BestFit program (version 9.0) from the University of Wisconsin Genetics Computer Group (Madison, WI).
Acknowledgments
We thank Dr. Kelley Moremen for generously providing human cDNA, Dr. Morag Park for the placenta cDNA library, Dr. Barbara Beatty for fluorescence in situ hybridization chromosomal analysis and Dr. Akio Miyabe for providing the P.pastoris strain DIPY115. We also thank Ariadni Athanassiadis, Homa Assar, Barry Sleno, and Peng Pang for technical assistance, Francesco Lipari for the yeast [alpha]1,2-mannosidase, and Dr. Pedro Romero for assistance with HPLC. This work was supported by an operating grant from the Medical Research Council of Canada, and L.O.T. and N.C.D. were recipients of a scholarship for graduate studies from the Fonds de la Recherche en Santé du Québec and the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
Abbreviations
ER, endoplasmic reticulum; RT-PCR, reverse transcriptase-polymerase chain reaction; ORF, open reading frame; YPD, yeast peptone dextrose; BMGY, buffered glycerol-complex; BMMY, buffered methanol-complex; HPLC, high-performance liquid chromatography.
References
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