©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of Bovine -Mannosidase (*)

(Received for publication, September 27, 1994; and in revised form, December 9, 1994 )

Hong Chen Jeffrey R. Leipprandt Christine E. Traviss Bryce L. Sopher Margaret Z. Jones Kevin T. Cavanagh Karen H. Friderici (§)

From the Department of Pathology, Michigan State University, East Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Deficiency of lysosomal beta-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans. A cDNA coding for the entire beta-mannosidase protein is described. Mixed oligonucleotides derived from bovine beta-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library. Clones covering about 80% of the C-terminal region were recovered. The missing 5`-region was obtained using the technique of 5`-rapid amplification of cDNA ends. The composite cDNA contains 3852 nucleotides, encoding 879 amino acids. The N-terminal methionine is followed by 16 amino acids displaying the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrates a single 4.2-kilobase transcript in various tissues from both normal and affected goats and calves. The mRNA level is decreased in tissues of affected beta-mannosidosis animals. The gene encoding beta-mannosidase is localized to human chromosome 4 as shown by Southern analysis of rodent/human somatic cell hybrids. This is the first report of cloning of lysosomal beta-mannosidase.


INTRODUCTION

Lysosomal beta-mannosidase (EC 3.2.1.25) is an exoglycosidase that cleaves the single beta-linked mannose residue from the nonreducing end of all N-linked glycoprotein oligosaccharides. Deficiency of beta-mannosidase activity results in an autosomal recessive inherited disorder, beta-mannosidosis. This lysosomal storage disease was first described in Nubian goats (1, 2, 3, 4) and more recently has also been found in humans(5, 6, 7, 8, 9, 10, 11, 12) and cattle(13, 14, 15) . Affected goats and cattle have very similar clinical features which include inability to stand, facial dysmorphism, intention tremors, and pastern joint hyperextension (14, 15, 16) . Deafness is a consistent finding in affected goats but not in newborn calves. Affected animals usually die in the neonatal period if intensive care is not provided. Widespread cytoplasmic vacuolation and dysmyelination in the central nervous system are characteristic lesions (17, 18) . Affected goats and calves are hypothyroid, possibly accounting for the central nervous system hypomyelination(19, 20) . The affected ruminants display a profound deficiency of beta-mannosidase activity in plasma and various tissues(4, 13, 21) . The primary storage products associated with the enzyme deficiency are the trisaccharide Manbeta1-4GlcNAcbeta1-4GlcNAc and lesser amounts of the disaccharide Manbeta1-4GlcNAc(2, 22) .

In contrast with the ruminant beta-mannosidoses, the human cases have a milder clinical expression and exhibit considerable heterogeneity (5, 6, 7, 8, 9, 10, 11, 12) . Clinical expression ranges from mild peripheral neuropathy and depression (12) to dysmorphology, mental retardation, and speech and hearing defects(5, 7, 8) . In human beta-mannosidosis the major accumulated product is the disaccharide (Manbeta1-4GlcNAc)(23, 24) . Presumably the variability and severity of the mutations responsible for inactivation of beta-mannosidase account for some of the phenotypic variation in the human cases. It is not known if the differences in disease expression between ruminants and humans is primarily related to the types of mutations, species differences in development, the nature of the storage products, or effects on thyroid function.

In order to understand the molecular lesions underlying beta-mannosidosis and the basis of the variation in disease expression between species, cloning and characterization of normal beta-mannosidase was initiated. Lysosomal beta-mannosidase is expressed at very low levels in most tissues and purification of this enzyme proved difficult (25, 26, 27, 28, 29) . The production of anti-beta-mannosidase monoclonal antibody permitted the establishment of a four-step chromatography procedure resulting in high purification levels of bovine beta-mannosidase in our laboratory(28, 29) . Peptide sequence analysis of the purified protein yielded 12 informative peptide sequences. Using this peptide sequence information we successfully isolated bovine beta-mannosidase cDNA clones covering the entire coding region, the 3`-noncoding region, and some of the 5`-noncoding region. This is the first report of cloning of lysosomal beta-mannosidase cDNA.


EXPERIMENTAL PROCEDURES

Partial Amino Acid Sequencing

beta-Mannosidase protein was purified from 2.4-kg bovine kidney (Ada Beef Co., Ada, MI) using anti-beta-mannosidase monoclonal antibody as described(28, 29) . The purity of the protein preparation after the Mono S step was verified by Coomassie Blue staining and Western analysis. The purified protein was dialyzed against 5 mM ammonium bicarbonate solution, lyophilized, and submitted to the Keck Foundation Biotechnology Resource Laboratory at Yale University for amino acid sequencing. Approximately 740 pmol of purified protein (from a total of 1240 pmol) was subjected to CNBr/trypsin digestion. Peptides were separated by C18 reverse phase HPLC (^1)and repurified by C8 reverse phase HPLC if needed. Sequence information was obtained from 12 peptides.

Construction of Synthetic Oligonucleotide Probes

Mixed and unique oligonucleotides were synthesized on Applied Biosystems model 394 and 380 B DNA synthesizers in the Macromolecular Structural Facility at Michigan State University. Regions with minimal codon redundancy were chosen to construct mixed oligonucleotide probes. Guessmers were constructed according to the typical codon usage frequency of human protein(30) . Gene-specific oligonucleotides for polymerase chain reaction (PCR) analysis were designed based on the Primer Program (Scientific & Educational Software). Oligonucleotides used in the screening and PCR analysis are listed in Table 1.



Labeling Probes

Oligonucleotide probes were prepared by 5` end labeling. Each 20-µl reaction contained 10 pmol of oligonucleotide, 15 pmol of [-P]ATP (6000 Ci/mmol, 10 mCi/ml, DuPont NEN), 1 µl of 20 times buffer (Boehringer Mannheim), and 10 units of T4 polynucleotide kinase (Boehringer Mannheim) and was incubated at 37 °C for 1 h. Unincorporated nucleotides were removed using an Nuctrap push column (Stratagene) following the manufacturer's instructions. cDNA fragments were labeled by the random primed method (Boehringer Mannheim) using [alpha-P]dCTP (3000 Ci/mmol, Amersham).

cDNA Library Screening

Normal bovine thyroid tissue was supplied to Clontech (Palo Alto, CA) for construction of a ZAP II cDNA library. The bovine thyroid cDNA library, primed by both oligo(dT) and random primers and consisting of 1.2 times 10^6 independent clones with an average insert size of 2.0 kb, was plated at a density of 1 times 10^4 plaque forming units/150-mm Petri dish. In situ amplification of plaques was performed as described (31) . The filters were prehybridized for 2 h and hybridized for 2-3 days at 46-48 °C in a solution containing 3 M trimethylammonium chloride (Aldrich or Sigma), 0.1 M sodium phosphate buffer, pH 6.8, 1 mM EDTA, pH 8.0, 5 times Denhardt's solution (1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin), 0.6% SDS, 100 µg/ml denatured herring sperm DNA (Boehringer Mannheim). Approximately 1-2 times 10^6 cpm/ml of 5` end-labeled mixed oligonucleotide probes with specific activity of 2-10 times 10^6 cpm/pmol were used during the hybridization. Filters were washed by standard procedures (31) and exposed to Kodak X-Omat AR film (Eastman Kodak Co.) at -80 °C for 1-3 days with intensifying screens. To maximize the screening, four 17-mer mixed oligonucleotides were used sequentially for probing the filters. Previous probes were removed by incubating hybridized filters in 0.4 M NaOH for 30 min at 45 °C and then 0.1 times SSC solution containing 0.1% SDS and 0.2 M TrisbulletCl, pH 7.5, for 30 min at 45 °C. Putative positive plaques were purified through several rounds of rescreening at lower densities and then excised as pBluescript plasmids according to the manufacturer's instructions. To isolate a full-length cDNA, the 1.6-kb insert of clone 47MJ4 was isolated and labeled to a specific activity of 1.4 times 10^9 cpm/µg. Up to 1 times 10^6 cpm/ml of the denatured probe was added in a 5 times SSPE (1 times SSPE, 0.15 M NaCl, 0.01 M NaH(2)PO(4)bulletH(2)O, 1.0 mM EDTA, pH 7.4) hybridization solution containing 50% formamide (Boehringer Mannheim), 0.5% SDS, 5 times Denhardt's solution, 10 µg/ml denatured herring sperm DNA to reprobe original filters at 42 °C. After approximately 20 h incubation, the filters were washed in 2 times SSC, (1 times SSC, 0.15 M NaCl, 0.015 M Na citrate, pH 7.0) 0.1% SDS, 2 times 10 min at room temperature, then washed until a low background signal was achieved. The final wash was 0.1 times SSC, 0.1% SDS solution at 65 °C for 30 min.

cDNA Synthesis and Polymerase Chain Reaction

Total RNA (10 µg) or poly(A) mRNA (1 µg) was heated at 65 °C for 5 min, then incubated with 10 µl of 5 times reverse transcription buffer (Life Technologies, Inc.), 5 µl of 0.1 M dithiothreitol, 5 µl of 10 mM dNTP mixture, 1.5 µl of 40 units/µl RNasin (Promega), 5-75 pmol of antisense oligonucleotide, and 200-400 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) in 50 µl of reaction mixture at 37 °C for 1 h. Synthesized first strand cDNAs were precipitated by adding an equal volume of 4 M ammonium acetate and 2 volumes of ethanol and resuspended in 50 µl of distilled water. An aliquot (1-2 µl) of the cDNAs was amplified in 50 µl reaction volume by AmpliTaq DNA polymerase (Perkin-Elmer Cetus) in the GeneAmp PCR system 9600 (Perkin-Elmer). Generally, 30-35 PCR cycles were performed and each PCR cycle consisted of 45 s denaturation at 94 °C, 1-min annealing at 46-55 °C, and 1-min extension at 72 °C. A 5-min predenaturation at 95 °C and an additional 10-min extension at 72 °C were applied before and after the cycle reactions, respectively. One-fourth of the amplified product was analyzed by electrophoresis on Nusieve 3:1 agarose gels (FMC BioProducts). To perform reamplification and nested PCR, agarose containing the band of interest was removed using a capillary tube. The agarose was either used directly (5 µl) in reamplification reactions or diluted in distilled water, and aliquots of agarose suspension were used in PCR reactions.

To analyze putative clones, PCR was performed using either plasmid DNA or crude phage lysates as templates. The crude phage lysates were prepared by adding an equal volume of 0.1 M NaOH to an aliquot of phage stock, incubating for 10 min at 95 °C, and then neutralizing by adding 1/20 volume of 2 M TrisbulletCl, pH 7.5.

To clone the 5`-region of beta-mannosidase cDNA, the 5`-RACE system kit (Life Technologies, Inc.) was used according to the manufacturer's instructions. Approximately 0.5 µg of poly(A) mRNA from bovine thyroid was copied into single strand cDNAs using a gene-specific primer MJ100 designed from the antisense strand of the 5` end of clone 17MJ48 (Table 1). The cDNA was tailed with homo-poly(dC) and then amplified by an anchor primer (AP) (Life Technologies, Inc.) and a nested gene-specific primer, MJ101. The 950-bp product was gel purified and reamplified by a universal amplification primer and the primer MJ101 and by universal amplification primer and a nested degenerate primer MJ48. The specific PCR products were gel purified and directly sequenced by a nested antisense primer MJ110 and cloned into the pCR II vector using a TA Cloning system (Invitrogen). Clones containing the correct insert size were subjected to sequencing.

RNA Isolation and Northern Blot Hybridization

Total RNA was extracted from various bovine and caprine tissues and from both normal and affected animals according to standard procedures(31) . Poly(A) RNA was isolated using a poly(A) quick mRNA kit (Stratagene). Poly(A) RNA samples were analyzed by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde as described (31) and blotted on Hybond-N membrane (Amersham). Northern hybridization was performed by standard procedures (32) using radiolabeled 47MJ4 insert, or the EcoRI fragment of 17MJ48 and a PCR product from 46MJ4 as probe(s). Filters were washed in a stepwise fashion according to the background signal with a final wash in 0.5 times SSC, 0.1% SDS for 30 min at 42 °C. The filters were exposed at -80 °C for 10 days. After removal of the beta-mannosidase probe, the blot was rehybridized to a cDNA probe of rat glyceraldehyde-3-phosphate dehydrogenase to normalize the amounts of RNA.

DNA Sequencing and Computer Analysis

Double strand DNA was prepared using a Magic (Wizard) miniprep DNA purification system (Promega). Sequencing was carried out by the Taq cycling method using either dye terminators or dye primers (M13 -21 primer and M13 reverse primer) on a 373A DNA sequencing system (Applied Biosystems) in the DNA Sequencing Facility at Michigan State University. The entire inserts in clones 46MJ4, 47MJ4, 17MJ48, and C2U48 were sequenced from both strands using internal oligonucleotide primers. PCR products were either directly sequenced after purification or were cloned into pCR II vector using the TA cloning kit. Plasmid DNAs were then prepared and sequenced as described above. DNA sequencing analysis and homology search against GenBank were performed using GCG program version 7, April, 1991 (Genetics Computer Group DNA Sequence Analysis Software).

Southern Hybridization Analysis

For chromosomal localization studies, a DNA panel of 24 human/rodent somatic cell hybrids was obtained from NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research (Camden, NJ). Approximately 15 µg of DNA of each hybrid was digested with PstI and separated on a 1% agarose gel. A PCR product of clone 46MJ4 was labeled by random priming to a specific activity of 4 times 10^8 cpm/µg and used as a probe in the Southern blot analysis. Hybridization was carried out in 50% formamide, 6 times SSC, 5 times Denhardt's, 0.5% SDS, 100 µg/ml denatured herring sperm DNA for 20 h at 42 °C. The final wash was in 1 times SSC, 0.1% SDS for 1 h at 42 °C. The blot was exposed to Kodak X-Omat AR film at -80 °C for 3-10 days with intensifying screens.


RESULTS

Peptide Sequencing

Bovine beta-mannosidase was purified approximately 150,000-fold by a four-step chromatography procedure. The final protein preparation revealed two predominant peptides of about 100 and 110 kDa, with 100 kDa being the major peptide as judged by Coomassie Blue staining (Fig. 1). Both peptides represent beta-mannosidase as shown in previous studies(28, 29) , and as demonstrated by reaction with anti-beta-mannosidase polyclonal serum (data not shown). Previous studies in our laboratory suggested that the N terminus of beta-mannosidase protein was blocked. In addition, trypsin-cleaved peptides appeared to be relatively insoluble. Therefore, the purified protein was subjected to combined CNBr and trypsin digestions. Fractionation of the CNBr/tryptic digest yielded multiple peptides suitable for direct sequencing or sequencing after rechromatography (Fig. 2). Sequencing of 14 peptides including those repurified by reverse-phase HPLC resulted in a total of nine non-overlapping peptides with complete sequences (Table 2) plus additional peptides with incomplete or mixed sequences. Four peptide sequences (142r12, 151r72, 218r24, and 103) were found to match previous peptide sequences (1, 2, and 3 in Table 2) resulting from sequencing of CNBr peptides of a deglycosylated gel purified 86-kDa protein (corresponding to the 100-kDa glycosylated beta-mannosidase protein). Searches with all peptide sequences obtained revealed no significant homologies with existing proteins in the GenBank, SwissProt, or EMBL data bases.


Figure 1: Verification of beta-mannosidase purification. An aliquot (12 pmol) of purified beta-mannosidase protein was fractionated on 7.5% SDS-PAGE (lane 2) and visualized by Coomassie Blue staining. High range (Bio-Rad) SDS-PAGE standard (lane 1).




Figure 2: Reverse phase HPLC profile of CNBr/tryptic-cleaved peptides of beta-mannosidase. Approximately 750 pmol of beta-mannosidase protein purified by a four-step column purification procedure were subjected to CNBr and trypsin digestions and separated by C18 reverse phase HPLC. Peptides 103, 171, 180, and 253 were sequenced directly. Mixed sequences were obtained from peptide 253. Peptides 104, 142, 151, 169, 218, 251, 265, and 267 were subjected to repurification by C8 reverse phase HPLC before sequencing. Peptides 251, 265, and 267 did not yield sequence.





Isolation and Characterization of cDNA Clones

Previous attempts at cloning beta-mannosidase cDNA included immunoscreening using anti-beta-mannosidase polyclonal antibodies(28, 29) , plaque hybridization using several mixed oligonucleotides or guessmers derived from peptide sequences, and PCR with various combinations of degenerate oligonucleotide primers. All these attempts were performed without success, presumably because of the very low level of expression of this lysosomal enzyme. The following strategy was thus adopted. First, to enrich for beta-mannosidase transcripts, bovine thyroid gland, the tissue showing the highest expression of beta-mannosidase activity, was used to construct a cDNA library. Second, oligonucleotides that, in previous studies, hybridized well with closely matching nucleotide sequences or that were derived from peptide sequences produced by two different sequencing sources (e.g. 142r12) were used for screening. Third, in an effort to increase detection levels, in situ amplification of plaques was performed(31) . Finally, multiple oligonucleotides and sequential hybridization were used.

By screening approximately 5 times 10^5 phage from the bovine thyroid cDNA library sequentially with four different degenerate oligonucleotides, MJ4, MJ48, MJ63, and MJ64, a total of 19 positive clones were detected. Of the 19 positive clones, three clones identified with MJ4 (43MJ4, 46MJ4, and 47MJ4) also hybridized with guessmers MJ7, MJ23, and MJ65. These oligonucleotides correspond to three different nonoverlapping peptides, suggesting clones 43MJ4, 46MJ4, and 47MJ4 are likely to be genuine positive clones. These three clones were plaque purified, excised as pBluescript plasmids, and subjected to further analyses. Restriction enzyme digestion showed that clones 43MJ4 and 47MJ4 each contained a 1.6-kb insert with identical restriction maps, indicating that they were the same clone, while clone 46MJ4 contained an insert of 1.8 kb with a restriction map similar to the other two clones (Fig. 3). Sequence analysis revealed that clones 43MJ4 and 47MJ4 (referred to as 47MJ4 in all later sections), and 46MJ4 all started at the same 5`-nucleotide which corresponded to a cleaved internal EcoRI site and contained an open reading frame of 730 bp. There were two single nucleotide differences between clones 46MJ4 and 47MJ4: C at position 2418 was replaced by G in clone 47MJ4 with no impact on the amino acid sequence, while C at position 2125 was substituted by T in clone 47MJ4 (Fig. 4), resulting in an amino acid change (H709D). The aspartic acid (D) was found in that position in the direct peptide sequence of peptide 151r72. The sequence homology between clones 46MJ4 and 47MJ4 diverged at 1182 bp from their 5` ends. The authenticity of both clones was established by colinearity of the predicted amino acid sequence of the two clones with five microsequenced peptide sequences (103, 218r24, 151r72, 180, and 171) (Fig. 4).


Figure 3: Restriction map and sequencing strategy for beta-mannosidase cDNA clones. Clones 17MJ48, 46MJ4, and 47MJ4 were obtained by oligonucleotide screening; r20, r8, and r2 were obtained by rescreening with 47MJ4; C2U48, a clone of the 5`-RACE product; PCRMJ82/74, a PCR product generated using primers from clones 17MJ48 and 46MJ4 sequence information; solid bar, coding region; double line and dash line, no homologies with 46MJ4; K, KpnI; B, BamHI; C, ClaI; E, EcoRI; X, XbaI; S, SalI; H, HincII; p, PstI; P, PvuII. A, poly(A) tail.






Figure 4: Nucleotide and deduced amino acid sequences of beta-mannosidase cDNA. Nucleotides upstream of the predicted initiation codon ATG are given negative numbers. Potential N-glycosylation sites are indicated by an asterisk (*). Colinear CNBr/tryptic peptides are underlined. Residues which do not match the peptide sequences determined by microsequencing are marked with [ ]. Two possible polyadenylation sites are underlined. Signal peptide sequence is double underlined. The arrow indicates the predicted signal peptide cleavage site.



To isolate a full-length cDNA, the 1.6-kb insert of clone 47MJ4 was gel purified, labeled, and used to probe the original filters. Three additional clones (r2, r8, and r20) were identified (Fig. 3). EcoRI digestion of plasmid DNAs indicated clone r20 lacked one of the EcoRI sites in the cloning site and contained an insert of approximately 1.4 kb. The insert size of clone r8 was close to 1.8 kb. Clone r2 appeared to have a large insert of approximately 4.3 kb, which was confirmed by Southern hybridization of EcoRI-digested plasmid and phage DNA. Analysis of these clones by PCR indicated that clone r2 contained approximately 1.2 kb more sequence in the 5` end than the existing clones. The r8 and r20 clones appeared to also start at the same internal EcoRI site as clones 46MJ4 and 47MJ4 (Fig. 3). Their 3` end sequences were nearly identical to that of clone 46MJ4, with clone r8 containing 20 additional base pairs including a short poly(A) tail (Fig. 3). Clone r2 encompassed most of the sequence of clone 46MJ4. However, the sequence homology diverged at 86 bp upstream of the 3` end of clone 46MJ4, and a long stretch of poly(A) tail was present in the 5` end. Furthermore, no open reading frame was found before the EcoRI site and the internal sequence homology with the 5` end of clones 46MJ4 and 47MJ4 ceased right at the EcoRI site of clone 46MJ4. These results clearly indicated that the EcoRI sites of these clones were not methylated by EcoRI methylase during the construction of this bovine thyroid cDNA library. Additional cloning artifacts occurred with clone r2.

The discovery of the failure of EcoRI methylation led us to re-evaluate two clones which had been identified previously by a mixed oligonucleotide probe (MJ48) in the initial screening. The peptide sequence corresponding to probe MJ48 was not found in the initial clones obtained, thus clones identified by MJ48 might correspond to sequences upstream of the cleaved EcoRI site. If their internal EcoRI site(s) was cleaved due to inefficient EcoRI methylation, these clones would not cross-hybridize with the three guessmers MJ7, MJ23, and MJ65, which were all located downstream of the EcoRI site. To evaluate these clones, PCR was performed on crude phage lysates and plasmid DNA from clones 9MJ48 and 17MJ48 using vector primers (M13 forward or reverse primer) and oligonucleotide primers for peptides not found in the original clones. Gene-specific PCR products were produced from clone 17MJ48. Sequence analysis showed that clone 17MJ48 contained an insert of 1119 bp and encoded 373 amino acids. Four additional peptide sequences (169r64, 169r65, 169r61, and 142r12) were found to match exactly with the predicted amino acid sequence of clone 17MJ48 (Fig. 4). This clone contained an internal EcoRI site and ended at an EcoRI site that had no linker sequences (Fig. 3). To show that clones 46MJ4 and 17MJ48 were continuous, PCR primers designed from each clone were used to produce the predicted amplification products from cDNA initiated by a downstream primer (Fig. 3, PCRMJ82/74).

Clone 17MJ48 plus clone r8 yielded a cDNA construct of about 3 kb. However, Northern hybridization indicated that the mRNA was 4.2 kb. The missing 5` end of the beta-mannosidase gene was obtained using 5`-rapid amplification of cDNA ends (RACE) (``Experimental Procedures''). The discrete 950-bp product produced by the RACE procedure was cloned and sequenced. Besides the expected peptide 142r12, also present in the 5` end of clone 17MJ48, an additional peptide sequence (104r86) was identified in the deduced amino acid sequence of the 5`-RACE products. The sequence of the 5`-RACE products also revealed a possible translation initiation codon 75 nucleotides from the 5` end, followed by an open reading frame. The nucleotides flanking the ATG (ACCATGC) were in good agreement with the consensus sequence for the eukaryotic initiation codon: A/GCCATGG(33) . Furthermore, the 16 amino acid residues following the initiation codon exhibited features characteristic of a signal sequence(34) , i.e. a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.

Northern Blot Analysis

A single transcript of approximately 4.2 kb was observed in normal and affected tissues from both cattle and goats using the cloned cDNA as probes (Fig. 5). The amount of transcript in affected tissues appeared to be significantly decreased compared to their normal counterparts. Thyroid tissue showed the highest level of beta-mannosidase mRNA.


Figure 5: Northern hybridization analysis of normal tissues and affected animals. Panel A, poly(A) RNA samples isolated from various bovine and caprine tissues were hybridized to a cDNA probe generated by PCR of clone 46MJ4 using primers MJ66 and MJ5 and an EcoRI fragment of clone 17MJ48. The blot was hybridized for 2 days and washed finally in 0.5 times SSC, 0.1% SDS at 42 °C for 30 min. The film was exposed for 10 days at -80 °C. Lane 1, affected bovine thyroid; 2, normal bovine thyroid; 3, affected bovine kidney; 4, normal goat liver; 5, normal goat kidney; 6, affected goat kidney. Panel B, rehybridization to a rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe after removal of the beta-mannosidase probe.



Southern Genomic Blot Analysis

Southern hybridization of a zoo blot showed that digestion of DNA with 6-hitter restriction enzymes produced 1-2 fragments in goat, human, or rodent and 2-5 fragments for bovine DNA. Human DNA showed single bands with high stringency wash and produced a heavy smear background at lower washing stringencies (data not shown).

To determine the human chromosomal location of beta-mannosidase, a panel of 24 human/rodent hybrid cell lines, each containing primarily a single human chromosome, was studied. Southern hybridization of PstI cleaved DNAs revealed a 1.7-kb band in the hybrid NA10115 (Fig. 6, lane 4) using a PCR product from the coding region of 46MJ4 as a probe. Its human origin was demonstrated by the observation of a band with the same size in the control human DNA. Ninety-seven percent of cells from the hybrid NA10115 contain human chromosome 4. Two bands of larger size were also found in several other hybrids (Fig. 6), probably due to incomplete restriction digestion. Like the zoo blot, the human control showed a smear background with the reduced stringency wash. beta-Mannosidase probes prepared from other regions of the cDNA also showed human specific bands only for chromosome 4 (data not shown).


Figure 6: Chromosome localization of beta-mannosidase cDNA. Approximately 15 µg of PstI-digested genomic DNA from 24 human/rodent somatic cell hybrids each containing a single human chromosome (indicated by lane number), were hybridized with a cDNA probe generated by PCR of plasmid DNA of clone 46MJ4 using primers MJ66 and MJ5. The hybridized blot was washed in 1 times SSC, 0.1% SDS for 1 h at 42 °C and exposed at -80 °C for 10 days. The number on top represents the human chromosome retained in that somatic cell line. H, human control DNA. (H), short exposure of lane H. M, mouse control DNA. C, Chinese hamster control DNA. S, DNA molecular marker III.




DISCUSSION

The entire coding sequence for lysosomal beta-mannosidase was determined from cDNA obtained by screening a bovine thyroid cDNA library with mixed oligonucleotides derived from the peptide sequence of purified protein and by PCR amplification using 5`-RACE. Three lines of evidence support the authenticity of this sequence. First, the amino acid sequence deduced from the nucleotide sequence is colinear with all beta-mannosidase peptide sequences (more than 100 residues) determined by direct amino acid sequencing. Second, the localization of the cDNA to human chromosome 4 is in agreement with previous reports(35, 36) . Third, the transcript of this cDNA in affected beta-mannosidosis animals is much lower than in normal animals.

The composite cDNA contains 3852 base pairs, consisting of a 74-bp 5`-noncoding region, followed by a 2640-bp coding region encoding 879 amino acids, then 1141-bp of 3`-noncoding region including a 13-bp poly(A) tail. The first in-frame ATG codon is flanked by a sequence in good agreement with the consensus sequence for eukaryotic translation initiation codons (33) and is followed by 16 amino acids containing the characteristic features of a signal peptide sequence(34) . Therefore, it is likely that the first in-frame ATG is the initiation codon for beta-mannosidase protein. Based on the(-3, -1) rule (34) we predict that the signal peptide is cleaved after residue 17. Since the beta-mannosidase protein is blocked at the N terminus, the precise cleavage site of the mature protein is unknown. Besides the signal peptide sequence, several other hydrophobic regions (e.g. amino acid residues 96-114 and 406-422) are revealed on a Kyte and Doolittle hydropathy plot(37) , however, none of them is likely to be a membrane spanning peptide.

Clones 46MJ4, r2, and 47MJ4 contained different 3`-regions (Fig. 3). The 3`-noncoding region of clone r8 probably represents the true sequence for the beta-mannosidase cDNA, since its sequence was found in clones 46MJ4 (only missing the final 20 bp) and r2 (except for the final 106 bp). The terminal sequences of the 3`-noncoding regions of clone 47MJ4 (approximately 450 bp) and r2 (approximately 1 kb) were not found in other clones and are likely to be cloning artifacts. Two possible poly(A) signal sequences (AATATA and ATTATA) were found in both 46MJ4 and r8, 32 and 14 bp upstream of the poly(A) tail. Various nonconsensus poly(A) signal sequences have been reported in several lysosomal enzymes and other mammalian genes(38, 39, 40) .

The peptide sequence deduced from the cDNA matches all peptide sequences obtained from purified beta-mannosidase, including those containing incomplete or mixed sequences. There are four discrepancies between the microsequenced amino acid sequences from CNBr/tryptic peptides and those predicted from the cDNA. Two (H686 and S864) occur at positions with uncertain residue assignments in peptide sequences and thus are most likely due to peptide sequencing artifacts. The other two (F65 and H709) may reflect natural polymorphisms.

Deglycosylation studies (29) suggested that bovine beta-mannosidase may contain seven to nine complex-type oligosaccharides. Only six potential glycosylation sites are present in the deduced amino sequence at residues 35-37, 77-79, 297-299, 302-304, 607-609, and 803-805. This difference might be due to an overestimation of protein size by SDS-PAGE, since attached carbohydrate can distort mobility(41) .

The 2586-bp coding region (after the removal of a 17-amino acid residue signal peptide) encodes 862 amino acids. This would give a predicted molecular mass of approximately 103 kDa. Lysosomal enzymes usually undergo limited proteolytic processing including N- or C-terminal trimming, or cleaving of internal peptides. An interesting feature in this cDNA is that a peptide sequence (171) is located immediately adjacent to the stop codon. If this peptide were derived from both 100- and 110-kDa proteins, then beta-mannosidase does not undergo C-terminal processing in kidney lysosomes. The region from amino acid residue 490 to residue 610 appears to be mainly hydrophilic and might be susceptible to proteolytic cleavage. With the complete coding sequence in hand, expression and pulse-chase studies will now be possible to determine both the biosynthesis and processing of beta-mannosidase and the relationship between the beta-mannosidase peptides observed during the protein isolation.

A single transcript of approximately 4.2 kb was observed in both normal and beta-mannosidosis animals and in both bovine and caprine tissues. The size difference observed between the cloned cDNA sequences of beta-mannosidase (3.85 kb) and the RNA transcript (4.2 kb) probably reflects some missing 5`-noncoding region and the poly(A) tail. The high GC content in the 5`-region might hinder reverse transcription in the 5`-RACE. The size of the transcripts was identical in goats and cattle as predicted by the observation that these ruminants had identical beta-mannosidase peptide sizes after deglycosylation(28, 29) .

No size difference was revealed between normal mRNA and beta-mannosidosis mRNA. However, the mRNA level in affected goats and calves was reduced compared to controls after normalizing for RNA loading with glyceraldehyde-3-phosphate dehydrogenase. The presence of normal sized mRNA in beta-mannosidosis animals implies that the beta-mannosidosis in ruminants is not caused by gene rearrangements or deletions. Reduction in mRNA levels could result from point mutations or small deletions producing a premature stop codon (42, 43, 44, 45) or mutations in the promoter region affecting transcription initiation. Southern analysis with several restriction enzyme digests revealed no gross gene rearrangements in affected and carrier beta-mannosidosis animals (data not shown). Hybridization of restriction enzyme-digested genomic DNA with a cDNA fragment of bovine beta-mannosidase revealed 2-6 bands of approximately 1-10 kb in the bovine species, two bands of approximately 2.8-7 kb in the caprine species, and a single band in humans. This result suggests that cattle and goats may have a large genomic structure with large introns, pseudogenes, or gene families. The genomic structure of human beta-mannosidase appears to be relatively small and not complex. Alternatively, the homology between ruminant and human beta-mannosidase may be limited to one or two exons. The reasons for the smear background observed in human DNA under a low stringency wash is unclear.

Homology searches against GenBank revealed no significant homologies between beta-mannosidase and other lysosomal enzymes. However, an unexpected striking homology to a human expressed sequence tag (EST01397) for an unknown gene from a human hippocampal cDNA library (46) was observed. There was 80% identity in a 454-bp region, from nucleotide 1720 to 2162 in the bovine cDNA. At the amino acid level, high homology to bovine protein was observed only in the central region of this human cDNA. The open reading frame in the human sequence is shifted by insertion of nucleotides at several positions, primarily in regions containing G stretches. Our ongoing cloning of human beta-mannosidase will verify the nature of this human tag sequence.

A beta-mannosidase gene had not previously been cloned from any species. The availability of the bovine beta-mannosidase cDNA will allow us to characterize the structure, regulation, and expression of this gene. It will also enable us to isolate beta-mannosidase genes from other species including human and goat and will facilitate the identification of molecular lesions underlying beta-mannosidosis in humans, goats, and cattle.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS16886 and by a grant from the Michigan State University development fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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) U17432[GenBank].

§
To whom correspondence should be addressed. Tel.: 517-353-9160; Fax: 517-432-1053.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s); RACE, rapid amplification of cDNA ends; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Susan Conrad (Michigan State University) for helpful discussion of the manuscript and Dr. Kenneth Williams (Yale University) for consultation on the peptide sequence analysis.


REFERENCES

  1. Hartley, W. J., and Blakemore, W. F. (1973) Acta Neuropathol. 25, 325-333 [Medline] [Order article via Infotrieve]
  2. Jones, M. Z., and Laine, R. A. (1981) J. Biol. Chem. 256, 5181-5184 [Abstract]
  3. Bole, P. J., Seaman, J. T., Gardner, I. A., and Bole, C. A. (1981) Bole. Vet. J. 57, 504-507
  4. Jones, M. Z., and Dawson, G. (1981) J. Biol. Chem. 256, 5185-5188 [Abstract]
  5. Wenger, D. A., Sujansky, E., Fennessey, P. V., and Thompson, J. N. (1986) N. Engl. J. Med. 315, 1201-1205 [Medline] [Order article via Infotrieve]
  6. Cooper, A., Sardharwalla, I. B., and Roberts, M. M. (1986) N. Engl. J. Med. 315, 1231
  7. Dorland, L., Duran, M., Hoefnagels, F. E. T., Breg, J. N., Fabery de Jonge, H., van Eeghen-Cransberg, K., Van Sprang, F. J., and van Diggelen, O. P. (1988) J. Inherited Metab. Dis. 11, Suppl. 2, 255-258 [Medline] [Order article via Infotrieve]
  8. Kleijer, W. J., Hu, P., Thoomes, R., Boer, M., Huijmans, J. G. M., Blom, W., van Diggelen, O. P., Seemanova, E., and Macek, M. (1990) J. Inherited Metab. Dis. 13, 867-872 [Medline] [Order article via Infotrieve]
  9. Cooper, A., Wraith, J. E., Savage, W. J., Thornley, M., and Noronha, M. J. (1991) J. Inherited Metab. Dis. 14, 18-22 [Medline] [Order article via Infotrieve]
  10. Poenaru, L., Akli, S., Rocchiccioli, F., Eydoux, P., and Zamet, P. (1992) Clin. Genet. 41, 331-334 [Medline] [Order article via Infotrieve]
  11. Wijburg, H., DeJong, J., Wevers, R., Bakkeren, J., Trijbels, F., and Sengers, R. (1992) Eur. J. Pediatr. 151, 311
  12. Levade, T., Graber, D., Flurin, V., Delisle, M.-B., Pieraggi, M.-T., Testut, M.-F., Carrière, J.-P., and Salvayre, R. (1994) Annu. Neurol. 35, 116-119 [Medline] [Order article via Infotrieve]
  13. Abbitt, B., Jones, M. Z., Kasari, T. R., Storts, R. W., Templeton, J. W., Holland, P. S., and Castenson, P. E. (1991) J. Am. Vet. Med. Assoc. 198, 109-113 [Medline] [Order article via Infotrieve]
  14. Bryan, L., Schmutz, S., Hodges, S. D., and Snyder, F. F. (1990) Biochem. Biophys. Res. Commun. 173, 491-495 [Medline] [Order article via Infotrieve]
  15. Jolly, R. D., Thompson, K. G., Bayliss, S. L., Vidler, B. M., Orr, M. B., and Bole, P. L. (1990) N. Z. Vet. J. 38, 102-105
  16. Jones, M. Z., Rathke, E. J. S., Cavanagh, K., and Hancock, L. W. (1984) J. Inherited Metab. Dis. 7, 80-85 [Medline] [Order article via Infotrieve]
  17. Jones, M. Z., Cunningham, J. G., Dade, A. W., Alessi, D. M., Mostosky, U. V., Vorro, J. R., Benitez, J. T., and Lovell, K. L. (1983) J. Neuropath. Exp. Neurol. 42, 268-285 [Medline] [Order article via Infotrieve]
  18. Lovell, K. L., and Jones, M. Z. (1983) Acta Neuropathol. 62, 121-126 [Medline] [Order article via Infotrieve]
  19. Boyer, P. J., Jones, M. Z., Nachreiner, R. F., Refsal, K. R., Common, R. S., Kelley, J., and Lovell, K. L. (1990) Lab. Invest. 63, 100-106 [Medline] [Order article via Infotrieve]
  20. Lovell, K. L., Jones, M. Z., Patterson, J., Abbitt, B., and Castenson, P. (1991) J. Inherited Metab. Dis. 14, 228-230 [Medline] [Order article via Infotrieve]
  21. Cavanagh, K., Dunstan, R. W., and Jones, M. Z. (1982) Am. J. Vet. Res. 43, 1058-1059 [Medline] [Order article via Infotrieve]
  22. Jones, M. Z., Rathke, E. J. S., Gage, D. A., Costello, C. E., Murakami, K., Ohta, M., and Matsuura, F. (1992) J. Inherited Metab. Dis. 15, 57-67 [Medline] [Order article via Infotrieve]
  23. Van Pelt, J., Hokke, C. H., Dorland, L., Duran, M., Kamerling, J. P., and Vliegenthart, J. F. G. (1990) Clin. Chim. Acta 187, 55-60 [Medline] [Order article via Infotrieve]
  24. Cooper, A., Hatton, C., Thornley, M., and Sardharwalla, I. B. (1988) J. Inherited Metab. Dis. 11, 17-29 [Medline] [Order article via Infotrieve]
  25. Kyosaka, S., Murata, S., Nakamura, F., and Tanaka, M. (1985) Chem. Pharm. Bull. (Tokyo) 33, 256-263 [Medline] [Order article via Infotrieve]
  26. Frei, J. I., Cavanagh, K., Fisher, R. A., Hausinger, R. P., Dupuis, M., Rathke, E. J. S., and Jones, M. Z. (1988) Biochem. J. 249, 871-875 [Medline] [Order article via Infotrieve]
  27. Iwasaki, Y., Tsuji, A., Omura, K., and Suzuki, Y. (1989) J. Biochem. (Tokyo) 106, 331-335 [Abstract]
  28. Sopher, B. L., Traviss, C. E., Cavanagh, K. T., Jones, M. Z., and Friderici, K. H. (1992) J. Biol. Chem. 267, 6178-6182 [Abstract/Free Full Text]
  29. Sopher, B. L., Traviss, C. E., Cavanagh, K. T., Jones, M. Z., and Friderici, K. H. (1993) Biochem. J. 289, 343-347 [Medline] [Order article via Infotrieve]
  30. Lathe, R. (1985) J. Mol. Biol. 183, 1-12 [Medline] [Order article via Infotrieve]
  31. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl, K. (1987) Current Protocols in Molecular Biology , Greene Publishing Associates and Wiley-Interscience (John Wiley & Sons), New York
  32. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  33. Kozak, M. (1986) Cell 44, 283-292 [Medline] [Order article via Infotrieve]
  34. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690 [Abstract]
  35. Fisher, R. A., Povey, S., Cavanagh, K., Dupuis, M., and Jones, M. Z. (1987) Am. J. Hum. Genet. 41, A165
  36. Lundin, L.-G. (1987) Biochem. Genet. 25, 603-610 [Medline] [Order article via Infotrieve]
  37. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  38. Stein, C., Gieselmann, V., Kreysing, J., Schmidt, B., Pohlmann, R., Waheed, A., Meyer, H. E., O'Brien, J. S., and vonFigura, K. (1989) J. Biol. Chem. 264, 1252-1259 [Abstract/Free Full Text]
  39. Stoltzfus, L. J., Sosa-Pineda, B., Moskowitz, S. M., Menon, K. P., Dlott, B., Hoopert, L., Teplow, D. B., Shull, R. M., and Neufeld, E. F. (1992) J. Biol. Chem. 267, 6570-6575 [Abstract/Free Full Text]
  40. Lin, B., Rommens, J. M., Graham, R. K., Kalchman, M., MacDonald, H., Nasir, J., Delaney, A., Goldberg, Y. P., and Hayden, M. R. (1993) Hum. Mol. Genet. 2, 1541-1545 [Abstract]
  41. Mahuran, D. J., Neote, K., Klavins, M. H., Leung, A., and Gravel, R. A. (1988) J. Biol. Chem. 263, 4612-4618 [Abstract/Free Full Text]
  42. Cheng, J., and Maquat, L. E. (1993) Mol. Cell. Biol. 13, 1892-1902 [Abstract]
  43. Mahuran, D. J. (1991) Biochim. Biophys. Acta Mol. Basis Dis. 1096, 87-94 [Medline] [Order article via Infotrieve]
  44. Zhang, Z.-X., Wakamatsu, N., Mules, E. H., Thomas, G. H., and Gravel, R. A. (1994) Hum. Mol. Genet. 3, 139-145 [Abstract]
  45. O'Dowd, B. F., Quan, F., Willard, H. F., Lamhonwah, A. M., Korneluk, R. G., Lowden, J. A., Gravel, R. A., and Mahuran, D. J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1184-1188 [Abstract]
  46. Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992) Nature 355, 632-634 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.