(Received for publication, September 27, 1994; and in revised form, December 9, 1994 )
From the
Deficiency of lysosomal -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
-mannosidase protein is described. Mixed oligonucleotides derived
from bovine
-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
-mannosidosis animals. The gene encoding
-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
-mannosidase.
Lysosomal -mannosidase (EC 3.2.1.25) is an exoglycosidase
that cleaves the single
-linked mannose residue from the
nonreducing end of all N-linked glycoprotein oligosaccharides.
Deficiency of
-mannosidase activity results in an autosomal
recessive inherited disorder,
-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
-mannosidase activity in plasma
and various tissues(4, 13, 21) . The primary
storage products associated with the enzyme deficiency are the
trisaccharide Man
1-4GlcNAc
1-4GlcNAc and lesser
amounts of the disaccharide
Man
1-4GlcNAc(2, 22) .
In contrast with
the ruminant -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
-mannosidosis the major accumulated product is the
disaccharide (Man
1-4GlcNAc)(23, 24) .
Presumably the variability and severity of the mutations responsible
for inactivation of
-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
-mannosidosis and the basis of the variation in disease expression
between species, cloning and characterization of normal
-mannosidase was initiated. Lysosomal
-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-
-mannosidase monoclonal antibody permitted the
establishment of a four-step chromatography procedure resulting in high
purification levels of bovine
-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
-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
-mannosidase cDNA.
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 TrisCl, pH 7.5.
To clone
the 5`-region of -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.
Figure 1:
Verification of -mannosidase
purification. An aliquot (12 pmol) of purified
-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 -mannosidase. Approximately 750
pmol of
-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.
By
screening approximately 5 10
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 -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 -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 -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.
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
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
-mannosidase probe.
To determine the
human chromosomal location of -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.
-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
-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
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.
The entire coding sequence for lysosomal -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
-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
-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 -mannosidase protein.
Based on the(-3, -1) rule (34) we predict that the
signal peptide is cleaved after residue 17. Since the
-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 -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 -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 -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 -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
-mannosidase and the relationship between the
-mannosidase
peptides observed during the protein isolation.
A single transcript
of approximately 4.2 kb was observed in both normal and
-mannosidosis animals and in both bovine and caprine tissues. The
size difference observed between the cloned cDNA sequences of
-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
-mannosidase peptide sizes after
deglycosylation(28, 29) .
No size difference was
revealed between normal mRNA and -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
-mannosidosis animals implies that the
-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
-mannosidosis animals (data
not shown). Hybridization of restriction enzyme-digested genomic DNA
with a cDNA fragment of bovine
-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
-mannosidase appears to
be relatively small and not complex. Alternatively, the homology
between ruminant and human
-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 -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
-mannosidase will verify
the nature of this human tag sequence.
A -mannosidase gene had
not previously been cloned from any species. The availability of the
bovine
-mannosidase cDNA will allow us to characterize the
structure, regulation, and expression of this gene. It will also enable
us to isolate
-mannosidase genes from other species including
human and goat and will facilitate the identification of molecular
lesions underlying
-mannosidosis in humans, goats, and cattle.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17432[GenBank].