From the
Cytidine monophospho-N-acetylneuraminic acid
(CMP-NeuAc) hydroxylase, which is the key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), has been purified from the
cytosolic fraction of mouse liver, as described in our previous paper.
The amino acid sequences of the purified CMP-NeuAc hydroxylase, and
peptides obtained by lysylendopeptidase digestion, were used to
synthesize specific oligonucleotide primers. A mouse cDNA clone of the
enzyme was obtained by a combination of the polymerase chain reaction
and rapid amplification of cDNA ends. The sequence of the clone
contained an open reading frame coding for a protein of 577 amino acids
with a predicted molecular mass of 66 kDa. The deduced sequence
included the amino acid sequences obtained for the purified enzyme and
peptides, and a complete match was obtained for 159 residues. The
enzyme has neither a signal peptide sequence nor a membrane spanning
domain, which is consistent with localization of the enzyme in the
cytosol. Transfection of a cDNA construct to COS-1 cells increased the
enzyme activity and the amount of NeuGc. Comparison of the sequence
with GenBank data indicated that no similar sequence has been reported
so far. Northern blot analysis of various mouse tissues with the enzyme
cDNA as a probe indicated that expression of NeuGc is related to the
level of CMP-NeuAc hydroxylase mRNA. On Southern blot analysis with the
same probe, cross-hybridizing bands were detected in the human and fish
genomes.
Cell membrane sialic acid is involved in cell-cell (1) and cell-pathogen interactions (2, 3) and in
binding of cells to extra cellular matrix(4) . Sialic acid is a
generic designation used for N-acylneuraminic acids and their
many derivatives(5, 6) . N-Acetylneuraminic
acid (NeuAc) and N-glycolylneuraminic acid (NeuGc)
The ratio of NeuGc to NeuAc in glycoconjugates varies among animal
species (16, 17) and among tissues of a single
species(18, 19, 20) . The regulation of NeuGc
and CMP-NeuGc expressions in mouse and rat tissues was suggested to be
dependent on the level of CMP-NeuAc hydroxylase
activity(14, 19) . NeuGc is barely detectable in the
brain of most mammals, although the sialic acid content of brain is
quite high(21) . Polysialic acids of N-CAM and gangliosides,
which are regarded to be functionally important in neural tissues,
contain NeuAc and not NeuGc. Thus, brain-specific suppression of NeuGc
is evident but its functional role is unknown. Normal chicken and human
tissues do not contain NeuGc. The occurrence of NeuGc in human T cells
activated in vitro (22) and various types of human cancer
tissues (23, 24, 25, 26, 27) was
demonstrated by immunological methods. Although the occurrence of NeuGc
remains to be confirmed by chemical methods, it is possible that NeuGc
would be an excellent cancer-associated antigen or tumor marker.
The
specialized physiological functions of NeuAc and NeuGc are not clear at
present. Influenza virus hemeagglutinins bind glycoconjugates that
contain NeuAc and NeuGc with different affinities, and the neuraminic
acids may play a role in determining susceptibility to viral
infection(28) . A cell adhesion molecule on marginal zone
macrophages, sialoadhesin, preferentially recognizes NeuAc-Gal-GalNAc
structure but not NeuGc-Gal-GalNAc(29) . These examples suggest
that sialic acid difference is biologically important in recognition
events mediated by carbohydrates.
To elucidate the mechanisms for
species- and tissue-specific and cancer associated regulations of NeuGc
expression, information about the structure of CMP-NeuAc hydroxylase is
required. Here we report the cDNA cloning of CMP-NeuAc hydroxylase,
analysis of its mRNA expression in mouse tissues, and detection of the
similar sequences in several animal species.
Hydropathy indexes by Kyte-Doolittle (32) were
estimated with a sliding window of nine amino acids.
Knowledge of the amino acid sequence of CMP-NeuAc hydroxylase
enabled us to design oligonucleotide primers and to generate cDNA that
encoded the entire protein and approximately 100 bp of both 5`- and
3`-untranslated regions. The structure of this enzyme is not similar to
other known hydroxylases, which is consistent with its unique substrate
specificity for CMP-NeuAc. Several sialyltransferases contain a
conserved amino acid sequence known as the ``sialylmotif,''
which may be a site that recognizes sialic acid(39) , but this
motif was not present in the CMP-NeuAc hydroxylase.
The enzyme was
purified from the cytoplasm(15) , but electron transport systems
are usually located in microsomal membranes. Alternatively, one might
suppose that the hydroxylase is a transmembrane protein or contains a
phosphatidylinositol-anchor and that it is artificially solubilized
during the purification by the action of proteinases or phospholipase
C. However, the nucleotide sequence of the hydroxylase obtained in this
study did not contain transmembrane domain and signal peptides for
membrane insertion, confirming the cytosolic origin of the enzyme. We
suggest the following models to explain how the soluble enzyme
interacts with the electron transport system. 1) soluble CMP-NeuAc
hydroxylase interacts for a short time with cytochrome b
The relationship between the level of mRNA of
CMP-NeuAc hydroxylase and the expression of NeuGc in glycoconjugates
was confirmed in several tissues. The inability of the brain to produce
NeuGc-containing glycoconjugates (18, 21) was evidenced
by the absence of the CMP-NeuAc hydroxylase mRNA (Fig. 3). Sialic
acid-containing glycoconjugates in the brain are considered to be
functionally important. For example, polysialylation of N-CAM reduces
homophilic interaction and cellular adhesion(1) ,
tetrasialoganglioside GQ1b potentiates neuritogenesis(41) , and
gangliosides exhibit cell type-specific
distribution(42, 43) . Interestingly, the sialic acids
of these functionally important sialoglycoconjugates are all NeuAc. It
is unclear at present why the expression of NeuGc should be suppressed
in the brain. Overexpression of the hydroxylase in the brain of
experimental animals could provide insight into this issue.
Although
the expression of NeuGc-containing glycoconjugates was detected in some
cancerous
tissues(23, 24, 25, 26, 27) ,
the existence of human CMP-NeuAc hydroxylase has not been documented
previously. The detection of a cross-hybridizing sequence in the human
genome suggests participation of CMP-NeuAc hydroxylase activation in
the tumor-associated expression of NeuGc-glycoconjugates. It will be
interesting to determine if hydroxylase mRNA can be detected in
cancerous tissues. A related fragment was also detected in a fish
genome. In some salmonid fish, the species-specific expression of
poly-NeuGc-glycoproteins in the eggs was reported(36) .
Therefore, investigation of the expression of the CMP-NeuAc hydroxylase
gene in salmonid fish eggs is also of interest.
CMP-NeuAc
hydroxylase, which regulates the overall velocity of CMP-NeuAc
hydroxylation, may be responsible for the species- and tissue-specific
expression of NeuGc-containing glycoconjugates(14) . Since the
synthesis of oligosaccharide structures is dependent upon not only the
level of glycosyltransferases, but also the amounts of the acceptor
oligosaccharides and donor nucleotide sugars, the activities of
glycosyltransferases do not always correlate well with the abundance of
oligosaccharide structures expressed in the cells. On the other hand,
CMP-NeuAc hydroxylase modifies the structure of the donor and may
directly regulate the expression of NeuGc in glycoconjugates. The
expression of NeuGc-glycoconjugates may also be regulated by factors
other than the hydroxylase itself, e.g. post-translational
modification of the enzyme, regulation of the accessibility of
cytochrome b
After 60 h, the transfectant and the mock transfectant were
harvested and subjected to the sialic acid analysis and the CMP-NeuAc
hydroxylase assay. CMP-NeuAc hydroxylase activities in the cytosolic
fractions were measured.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We are grateful to Drs. Kiyomitsu Nara, Hideo Kubo,
and Yoko Nadaoka of the Tokyo Metropolitan Institute of Medical Science
for the suggestions and technical support, and Dr. Hiroshi Kurata,
Yukiko Tamagawa, and Mitsuyo Ohkawa of Kyoto University for their
technical advice and support. The authors also wish to thank Prof.
Donald M. Marcus for revising the manuscript and Prof. Tamio Yamakawa
for his encouragement.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)are two of the most abundant derivatives, and both
may be further modified by O-acetylation. Some reports
suggested that NeuGc was produced from NeuAc through enzymatic
hydroxylation of the N-acetyl residue of free NeuAc,
CMP-NeuAc, or glycoconjugate-linked NeuAc(7, 8) , but
recent results have indicated that the major mechanism for biosynthesis
of NeuGc is hydroxylation of
CMP-NeuAc(9, 10, 11) . In addition, the
hydroxylation is carried out by an electron transport system, which
includes NADH-dependent cytochrome b
reductase,
cytochrome b
, and CMP-NeuAc hydroxylase. The
hydroxylase accepts electrons from cytochrome b
and catalyzes the terminal
reaction(12, 13, 14) . We have purified
CMP-NeuAc hydroxylase from the cytosolic fraction of mouse liver and
demonstrated that the enzyme is highly specific to CMP-NeuAc and does
not use free NeuAc or NeuAc-containing GM3 as a substrate(15) .
Protein Sequencing
Purification of CMP-NeuAc
hydroxylase was described previously(15) . An aliquot (37
µg) of the finally purified enzyme was lyophilized and then
dissolved in 40 µl of 8 M urea. After dilution with 120
µl of 0.1 M Tris-HCl buffer, pH 9.0, 0.4 µg of
lysylendopeptidase (Wako Pure Chemicals) was added, followed by
incubation at 37 °C overnight. The resulting peptide fragments were
separated by HPLC on a Wakosil 5C18 column (4 150 mm; Wako Pure
Chemicals). Elution was performed with a linear concentration gradient
of acetonitrile, 0-60%, in 0.1% trifluoroacetic acid, in 60 min.
Twelve major peaks were recovered and subjected to protein sequencing
using an Applied Biosystems 470A protein sequencer.
NH
-terminal amino acid sequencing was also performed using
6 µg of the purified hydroxylase.
Polymerase Chain Reaction (PCR) with Degenerate
Oligonucleotides
Degenerate oligonucleotides designed on the
basis of the amino acid sequences of the lysylendopeptidase-digested
peptides and the intact enzyme, as shown in Fig. 1A,
were synthesized with an Applied Biosystems 391 DNA synthesizer. Each
oligonucleotide primer contained either an EcoRI or HindIII recognition sequence at the 5`-end. A pool of cDNA was
prepared by reverse transcription with random primers using mouse liver
total RNA as a template. PCR was performed using sense and antisense
primers (125 pmol each), and 1.3 units of Taq DNA polymerase
(Promega) in a reaction mixture (25 µl) containing 1 µg of
cDNA(30) . Amplification was carried out by 30 cycles of 94
°C for 0.5 min, 37 °C for 1 min, and 72 °C for 1.5 min,
using a Zymoreactor II thermal cycler (Atto Corp.). The PCR products
were analyzed by 2% agarose gel electrophoresis and stained with
ethidium bromide.
Figure 1:
A, NH-terminal amino acid
sequence and partial amino acid sequences of
lysylendopeptidase-digested peptides a and b. The
sequences of the degenerate oligonucleotide primers (pr. 1, pr. 2, and a-pr. 1) used for the PCR experiments are
shown under the corresponding amino acid sequences (underlined). Boxes show the linker sequences
(GAATTC, EcoRI; and AAGCTT, HindIII). B,
amplification of cDNA fragments by PCR using the above mentioned
primers and newly synthesized completely matched primers (pr.
3, a-pr. 2, a-pr. 3, and a-pr. 4). Hooked arrows indicate the positions and directions of these
primers. The sequences of these primers are shown in Fig. 2. pr. 3 and a-pr. 2 have linker sequences TCTAGA (XbaI)
and CTCGAG (XhoI), respectively.
Isolation and Characterization of a cDNA
Clone
After being digested with appropriate restriction enzymes,
the PCR amplified fragments were subjected to electrophoresis, excised
from the gel, purified with a QIAEX gel extraction kit (Qiagen), and
then subcloned into Bluescript II KS (Stratagene). The
constructs were transfected into XL1-Blue strain. Clones with the
inserts were identified by color selection with
isopropyl-1-thio-
-D-galactopyranoside and
5-bromo-4-chloro-3-indolyl-
-D-galactoside, and plasmid
DNAs were isolated from Escherichia coli grown in small scale
cultures(31) . The sequencing reaction with a thermal cycler was
performed with Dye Deoxy terminators (Applied Biosystems) and Ampli Taq DNA polymerase (Applied Biosystems). The M13 universal
primer or several synthetic oligonucleotide primers were used. Gel
electrophoresis and analysis of data were performed with a 373A DNA
sequencer (Applied Biosystems). Three clones were sequenced to
compensate for misreading by Taq polymerase. In the case of
the 5`-non-coding sequence, the PCR amplified fragments were directly
sequenced, without subcloning, using specific oligonucleotides as
primers.
Expression of the cDNA in COS-1 Cells
To amplify
the entire coding region of CMP-NeuAc hydroxylase, two primers, each of
which contains either the 5`- or 3`-end of coding region and BamHI recognition sequence at the 5`-end, were synthesized.
Amplified fragments with mouse liver cDNA were digested with BamHI (Takara Shuzo Corp.) and inserted into a Bluescript II
vector. An insert, the sequence of which was confirmed to be the same
as the cloned sequence, was ligated into BamHI site of the
expression vector, pdKCR(33) . A construct of the correct
direction was amplified in a large scale, and purified by two cycles of
CsCl gradient centrifugation(31) . COS-1 cells obtained from the
Japanese Cancer Research Bank were grown in Dulbecco's modified
Eagle's medium (Nissui Pharmaceutical Co. Ltd.) with 10% fetal
calf serum (Life Technologies, Inc.) and kanamycin. Six dishes of COS-1
cells (5 10
cells/10 cm dish) were transfected with
the purified plasmid (20 µg/dish) by calcium phosphate
precipitation methods(34) . At 60 h of incubation, the cells
were harvested, washed, and collected by centrifugation. One-fifth of
the pelleted cells was subjected to the determination of sialic acid
species according to Hara et al.(35) . Briefly, the
pelleted cells were hydrolyzed in 0.9 ml of 0.05 N H
SO
at 80 °C for 3 h, and released
sialic acids were treated with 3.6 ml of 3. 5 mM of
1,2-diamino-4,5-methylenedioxybenzene in 1.7 M acetic acid at
50 °C for 2.5 h. NeuAc and NeuGc were determined by HPLC with a
TSK-gel ODS-80TM column (Tosoh). Four-fifths of the pelleted cells were
homogenized, and the cytosolic fraction was used for the determination
of protein concentration and CMP-NeuAc hydroxylase activity as
previously reported (14).
Northern Blot Analysis
Poly(A) RNA from mouse tissues was prepared using an ISOGEN acid phenol
procedure kit (Wako Pure Chemicals) and Oligotex dT
(Takara Shuzo Corp.), following the manufacturers'
protocols. Approximately 3 µg of poly(A)
RNA
samples was subjected to electrophoresis in a 1% agarose gel containing
formaldehyde and then transferred to a MagnaGraph nylon membrane filter
(Micron Separations Inc.). A radiolabeled probe (3
10
counts/min/µg) was prepared with a gel-purified cDNA
containing the whole coding region of CMP-NeuAc hydroxylase or 2.0-kb
human
-actin cDNA (Clontech) using a random primer DNA labeling
kit (Takara Shuzo Corp.). Hybridization was performed overnight at 42
°C in 50% formamide, 5
SSC (1
SSC, 150 mM NaCl, and 15 mM sodium citrate, pH 7.0), 50 mM sodium phosphate buffer, pH 7.0, 0.5% skim milk, 1.5% SDS, and 100
µg/ml yeast RNA. The blot was washed twice in 1
SSC and
0.1% SDS at 65 °C for 10 min, and then three times in 0.1
SSC and 0.1% SDS at 65 °C for 20 min. The bands were visualized and
quantified with a Fujix BAS 2000 Bio-imaging Analyzer (Fuji Photo
Film).
Southern Blot Analysis
A BIOS EVO Blot digested
with EcoRI was purchased from BIOS Laboratories. The amount of
DNA blotted is adjusted to assure the same copy number of the genome
available among seven species. Preparation of a radiolabeled probe was
performed in the same way as described for Northern blot analysis.
Hybridization was performed essentially as for Northern blot analysis
except that the proportion of formamide was decreased to 37.5%. After
overnight hybridization, the blot was washed with 1 SSC and
0.1% SDS at room temperature, 50 °C, and finally 65 °C. The
radioactivity was monitored with a Fujix BAS 2000 Bio-imaging Analyzer.
Discrete bands were visible only after the washing at 65 °C.
Protein Sequencing and Amplification of cDNA Fragments
by PCR
The amino acid sequences of the two major peptides and
the NH terminus are shown in Fig. 1A, and
these sequences were used for designing mixed oligonucleotide primers
for PCR. First, a fragment of 158 bp was amplified from mouse liver
cDNA with sets of degenerated primers designed for peptides a and b (pr. 2 and a-pr. 1,
respectively, in Fig. 1A). This fragment could not be
amplified with the cDNA prepared from mouse brain, which was used as a
negative control because it does not express CMP-NeuAc
hydroxylase(14) . The amplified fragment was subcloned into
Bluescript II KS
and then sequenced. Based on the
sequence, completely matched oligonucleotide primers (a-pr. 2 and pr. 3, see Fig. 1B and 2) were
synthesized for further experiments. The next PCR was performed between
pr. 1, designed for the NH
-terminal amino acid sequence,
and a-pr. 1. On this PCR, no specific band was visibly amplified, but
when an aliquot of the reaction product was diluted and subjected to a
second PCR (30) with a primer corresponding exactly to an inner
sequence (a-pr. 2) and pr. 1, a fragment of 1.5 kb pairs was
amplified. This fragment seemed to cover the whole coding region of the
enzyme except for the 5`- and 3`-ends, as judged on comparison of its
size with the molecular mass of the hydroxylase(15) . Since the
fragment had an inner EcoRI site, the PCR product was digested
with EcoRI, ligated into the EcoRI site of Bluescript
II KS
, and then sequenced. Antisense primers (a-pr. 3 and a-pr. 4) were synthesized for the
following experiments (see Fig. 1B and 2).
Rapid Amplification of the 5`- and 3`-cDNA
Ends
For amplification of the 5`-end of the cDNA, a template was
prepared by reverse transcription with a-pr. 3 and polyadenylation of
the 5`-end (30). A 650-bp fragment was amplified by PCR with oligo(dT)
and a-pr. 4, as shown in Fig. 1B, and a part of the
fragment was sequenced. The 3`-end of the cDNA was also amplified with
pr. 3 and oligo(dT), using a cDNA prepared by reverse transcription
with oligo(dT) as a primer. Approximately 250- and 700-bp fragments
were amplified. The 250-bp fragment and a part of the 700-bp fragment
were sequenced, and the sequences indicated that the 700-bp fragment
included the 250-bp fragment and both contained an 89-bp coding
sequence.
Nucleotide Sequence and Expression of CMP-NeuAc
Hydroxylase
The merged sequence of the overlapping cDNA
fragments indicated a single open reading frame, and about 100-bp 5`-
and 3`-untranslated regions. The nucleotide sequence (1731 bp) and the
deduced amino acid sequence (577 amino acids) for the open reading
frame are shown in Fig. 2. Although the 5`-untranslated region
has not been completely sequenced, stop codons were emerged at
-93, -59, and -46 in each of three frames. There are
adjoining methionines at the potential site for translation initiation.
The upper methionine was tentatively taken as an initiation site. The
first four amino acids at the NH terminus could not be
detected on sequencing of the purified hydroxylase, and thus these
amino acids were cleaved, possibly through post-translational
processing or artificial cleavage during the purification. All the
amino acid sequences of the 12 lysylendopeptidase-digested peptides and
NH
terminus of the purified enzyme, 159 amino acid residues
long in total, were found in the open reading frame of the deduced
amino acid sequence (underlined in Fig. 2). The
molecular mass calculated from the deduced amino acid sequence is 66
kDa, which is in good accordance with that determined by
SDS-polyacrylamide gel electrophoresis under reducing conditions (64
kDa)(15) . A search of GenBank data revealed no similar sequence
reported so far. A hydropathy plot (32) of the enzyme did not
identify a potential transmembrane region, suggesting that the enzyme
is translated on free ribosomes as a cytosolic protein.
Figure 2:
Nucleotide sequence and deduced amino acid
sequence of CMP-NeuAc hydroxylase. The sequences corresponding to the
NH terminus and the lysylendopeptidase-digested peptides of
the enzyme are indicated by underlines. There are two
potential sites for translation initiation. The upper Met is taken as
site 1 here. The sequences corresponding to the synthesized primers are boxed.
As shown in , the COS-1 cells transfected with the cloned cDNA
contained an increased amount of NeuGc, compared with a mock
transfectant. This change is confirmed to be due to the increase of
CMP-NeuAc hydroxylase activity. The results all togehter indicate that
the cloned cDNA encodes for mouse CMP-NeuAc hydroxylase.
Northern Blot Analysis
To test the possibility
that the expression of CMP-NeuAc hydroxylase regulates the
tissue-specific expression of NeuGc-containing glycoconjugates, mRNA
isolated from mouse brain, thymus, liver, kidney, and spleen was probed
at high stringency with a radiolabeled cDNA fragment. It was reported
that NeuGc was hardly detected in the brain gangliosides, whereas both
NeuAc and NeuGc were expressed in the other tissues(18) .
Consistent with this observation, 3.6 and 10 kb mRNA bands were
detected for all tissues except brain (Fig. 3). The intensity
ratio of 3.6 and 10 kb mRNA bands is almost the same among tissues
expressing NeuGc, and both bands are not detected in the brain. The
10-kb transcript, which is much longer than the cDNA we obtained, would
be a product of alternative splicing or use of alternative
polyadenylation signals in a 3`-untranslated region.
Figure 3:
Northern blot analysis of CMP-NeuAc
hydroxylase. Approximately 3 µg of poly(A) RNA
obtained from mouse liver (l), brain (b), thymus (t), spleen (s), and kidney (k) was
subjected to electrophoresis and probed at high stringency with a
radiolabeled cDNA containing the whole coding region of CMP-NeuAc
hydroxylase or 2.0-kb human
-actin cDNA. The positions and sizes
of marker RNAs are indicated at the right.
Southern Blot Analysis
As shown in Fig. 4,
human and fish genomic DNAs contain cross-hybridizing sequences,
whereas chicken, frog, lobster, and mussel DNAs were negative. These
data are quite interesting because expression of NeuGc was detected in
several human cancerous tissues (23, 24, 25, 26, 27) and in
polysialoglycoproteins of several kinds of fish (36) but not in
chickens or frogs(37) . Sialic acid have not been detected in
bivalves to date(38) .
Figure 4:
Genomic Southern blot analysis of various
animal species. Blots containing EcoRI digested genomic DNA of
man, mouse, chicken (Gallus domesticus), frog (Xenopus
laevis), lobster (Homarus americanus), mussel (Mytilus edulis), and a fish (Tautoga onitis) were
probed at low stringency with a radiolabeled CMP-NeuAc hydroxylase
cDNA. The positions and sizes of marker DNA (-HindIII
digest) are indicated at the left.
on microsomes, when the transfer of electrons occurs; or 2)
soluble forms of cytochrome b
and NADH-cytochrome b
reductase are involved in the reaction. At
present, the latter idea is unlikely because mRNA encoding only the
microsomal form of cytochrome b
was observed in
mouse liver(40) . To clarify this point, reconstitution
experiments mimicking the in vivo interaction between soluble
CMP-NeuAc hydroxylase and the membrane-bound electron transport system
are required.
and cytochrome b
reductases, reutilization of incorporated NeuGc or
NeuGc-glycoconjugates as a source of CMP-sialic acids(44) , and
involvement of sialyltransferases and CMP-sialic acid transporters
specific to either CMP-NeuAc or CMP-NeuGc, although there are no data
to support such specificity at present(45, 46) .
Modification of sialic acids from NeuAc to NeuGc by artificial
expression of CMP-NeuAc hydroxylase in cultured cells or animals is now
possible because of availability of cDNA. Further studies of the
enzyme, using the hydroxylase cDNA, will facilitate elucidation of
functions of NeuGc-containing glycoconjugates and the mechanisms of
NeuGc expression in ontogeny and phylogeny.
Table: Expression of cloned cDNA in COS-1 cells
/EMBL Data Bank with accession number(s)
D21826.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.