Molecular cloning of the cDNA encoding a murine sialic acid-specific 9-
O
-acetylesterase and RNA expression in cells of hematopoietic and non-hematopoietic origin
Molecular cloning of the cDNA encoding a murine sialic acid-specific 9- O -acetylesterase and RNA expression in cells of hematopoietic and non-hematopoietic origin
Angela
Stoddart
1,2,*
,
Yu
Zhang
1,3
and
Christopher J.
Paige
1,2
1
The Wellesley Hospital Research Institute,
2
Department of Immunology and
3
Department of Medical Biophysics, University of Toronto, 160 Wellesley Street
East,
Toronto
, Ontario M4Y 1J3,
Canada
Received June 21, 1996;
Revised and Accepted August 28, 1996
DDBJ/EMBL/GenBank accession nos U61183, X98625
ABSTRACT
We describe the isolation of a cDNA encoding a murine sialic acid-specific 9-
O
-acetylesterase as well as its expression pattern in cells of both
hematopoietic and non-hematopoietic origin. This enzyme catalyzes the removal of
O
-acetyl ester groups from position 9 of the parent sialic acid
N
-acetylneuraminic acid. The cDNA is 2105 nt in length and encodes a protein
of 541 amino acids with a predicted molecular weight of 61 kDa, not including
oligosaccharides linked to eight potential
N
-glycosylation sites. The cDNA encoding the acetylesterase displays a
widespread distribution in various cell lines and tissues. Expression studies
of B lineage cell lines and primary fetal liver cells revealed a
developmentally regulated expression pattern in cells of hematopoietic origin.
Given the importance of 9-
O
-acetylation of sialic acids, the cloning of the cDNA encoding a sialic
acid-specific 9-
O
-acetylesterase will be helpful in understanding further the regulation of
this post-translational modification and the biological consequences thereof.
INTRODUCTION
The sialic acids are a diverse family of nine carbon acidic sugars often found
as the terminal units of oligosaccharide chains on cell surface glycoconjugates
(
1
). Many of the naturally occurring modifications of the parent sialic acid,
N
-acetylneuraminic acid, arise from
O
-acetylation at the 4, 8 or, more commonly, the 7 and 9 positions (
2
,
3
). Since
O
-acetyl esters at the 7 position can undergo spontaneous migration to the 9 position under physiologic conditions, 9-
O
-acetyl-
N
-acetylneuraminic acid is the predominant acetylated form on cell surface glycoconjugates (
4
,
5
).
The 9-
O
-acetylation of sialic acids is regulated in a developmental- and tissue-specific manner in certain systems. For example, the 9-
O
-acetylated form of the disialoganglioside G
D3
is found only in specific regions of the developing nervous system and its
expression decreases soon after birth (
6
,
7
). These
O
-acetyl ester groups can affect several biological processes, including
virus binding, bacterial neuraminidase activity, lectin recognition and tumor antigenicity (
1
,
2
). Understanding the mechanisms that control 9-
O
-acetylation of sialic acids is therefore of broad interest.
Enzyme activities capable of removing
O
-acetyl groups from the 9 position of sialic acids have been described in certain mammalian
viruses (
8
-
10
), in human erythrocytes (
11
) and in murine and equine livers (
12
-
14
). Two distinct sialic acid-specific
O
-acetylesterases have been purified from rat liver Golgi-enriched preparations (
15
); a cytosolic sialate:9-
O
-acetylesterase (CSE) and a membrane-associated intralumenal sialate:9-
O
-acetylesterase (LSE).
Recent studies show that glycoproteins found on B lymphocytes also contain 9-
O
-acetylated sialic acids (
16
). In experiments designed to identify genes expressed at distinct stages of B
cell development, we have isolated a murine cDNA which encodes a protein whose
amino acids sequence shares identity with the rat sialic acid-specific
O
-acetylesterase (LSE). This gene is expressed in late, but not early, B cells, raising the possibility that regulation
of sialate:9-
O
-acetylation during B cell differentiation may have developmental
significance.
MATERIALS AND METHODS
Mice
C57BL/6 and CD1 mice were purchased from the Jackson Laboratory (Bar Harbor, ME)
and housed at the animal facility at the Wellesley Hospital Research Institute.
Timed matings were scheduled as previously described (
17
).
Cell lines
J558, WEHI-231, WEHI-3, EL4, L929 and P338D1 were purchased from the American Type
Culture Collection (ATCC). CB5 was obtained from Dr S.Benchimol (Ontario Cancer Institute, Toronto, Canada). BMS2.2 was provided by Dr P.W.Kincade (Oklahoma Medical
Research Foundation, Oklahoma City, OK). IIB4, CB17 1.1 and CB17 5.1 are
Abelson murine leukemia virus (A-MuLV)-transformed B lineage cell lines and were generated in our
laboratory. 70Z/3 is a pre-B cell line (
18
), WEHI-231 is an immature (sIgM
+
) B cell line and J558 is a myeloma cell line. RBL5 and EL4 are T cell lines,
WEHI3 and P338D1 are macrophage cell lines, CB5 is an erythroid cell line, 3T3
and L929 are fibroblast cell lines and BMS2.2 is a stromal cell line.
RNA preparation and Northern analysis
Total RNA was isolated from CD1 mouse tissues and cultured cell lines as
previously described (
19
). Poly(A)
+
RNA was selected by passage over oligo(dT)-cellulose (Pharmacia) (
20
). For Northern analysis, 5 [mu]g poly(A)
+
RNA were separated on 1% agarose gels containing 20 mM NaHPO
4
and 1 M formaldehyde, transferred to Hybond-N nylon membranes (Amersham), UV-immobilized and hybridized with
32
P-labeled probes prepared by a random hexamer-primed method (
21
). Hybridization was at 42oC in 5* SSPE, 2% SDS, 5* Denhart's solution, 100 [mu]g sheared/boiled salmon sperm DNA, 100 [mu]g poly(A) and 50% formamide. The membranes were
washed in 0.1* SSC/0.1% SDS at 65oC.
Differential display PCR
Differential display PCR was performed following the method described by Liang
and Pardee (
22
) with a GenHunter Kit (Brookline, MA). Poly(A)
+
RNA (0.2 [mu]g) from IIB4 and 70Z/3 cells was used for first strand cDNA synthesis with
each of the four modified oligo(dT) primers (T12MN). The first strand cDNA was
used as a template in the subsequent polymerase chain reaction (PCR), which contained 50 mM KCl, 1.5 mM MgCl
2
, 10 mM Tris-HCl, pH 8.3, 0.2 [mu]M 5'-arbitrary 10mer, 1 [mu]M T12MN, 2 [mu]M dNTPs, 12.5 [mu]Ci [
35
S]dATP (100 Ci/mmol), 1 U Taq DNA polymerase (Perkin Elmer). PCR was performed
as follows: 94oC, 30 s; 40oC, 2 min; 72oC, 30 s for 40 cycles. Four microliters of the PCR products from
the two cell lines were run side by side on a 6% acrylamide:urea sequencing
gel. The dried gel was exposed to X-ray film and the autoradiogram was analyzed for differentially displayed
bands. These were cut from the gel and the DNA was eluted by soaking the gel slices in 100 [mu]l Tris-EDTA (TE) buffer for 10 min, followed by boiling for 10 min. The eluted DNA was precipitated with glycogen and ethanol and resuspended in 10 [mu]l dH
2
O. This DNA was reamplified with the same combination of primers used in the
first PCR. The reamplified DNA was gel purified and used as a probe in Northern analysis to confirm
differential expression. The amplified DNA was then subcloned using the TA
Cloning Kit (Invitrogen, San Diego, CA).
cDNA library construction and screening
A 70Z/3 cDNA library was constructed essentially as described by Sambrook
et al.
(
20
). Five micrograms of poly(A)
+
RNA was reverse transcribed using an oligo(dT)12-18 primer. The mRNA-cDNA hybrid was treated with RNase H and the resulting mRNA
fragments served as primers for the synthesis of second strand cDNA. The double-stranded cDNA was made blunt-ended with Klenow fragment and then ligated to an
Eco
RI/
Not
I adapter. This adapter-tailed cDNA was purified to remove the unligated adapters, then inserted into the [lambda] ZAPII vector (Stratagene, La Jolla, CA). The constructs were
packaged into infectious [lambda] phage particles and amplified in
Escherichia coli
strain XL1-Blue. The ratio of recombinants in the library was >95% and the total
yield of the recombinants was 4 * 10
6
.
The size of cDNA inserts from 12 randomly picked up clones ranged from 0.8 to
4.5 kb, with an average of 1.4 kb.
The cDNA library was next screened with one of the differential display PCR
fragments, a 155 bp cDNA fragment designated 7a3. Ten positive clones were
isolated by three rounds of screening. The
in vivo
excision procedure was performed to release pBluescript plasmid from the [lambda] ZAPII vector. The insert size of the 10 clones ranged from 2.1 to 2.5
kb. The nucleotide sequence of each clone from both strands was determined by
the dideoxynucleotide chain termination method (
23
).
5
'
RACE of 7a3 mRNA
The 5'-end of the 7a3 cDNA was amplified by the 5' RACE (rapid amplification of complementary DNA ends) method
with the reagent kit from Clontech (Palo Alto, CA). The 3' (7a3 specific) primer was 5'-CAA AGT CTG TTG CGC CAT CAC TTC-3' and the 5' (AP1 primer) was 5'-CCA TCC TAA TAC GAC TCA CTA
GGG C-3'. The amplified PCR product was subcloned using the TA Cloning kit
(Invitrogen, San Diego, CA).
Isolation of bipotential B cell-macrophage progenitors
Liver cell suspensions were prepared from day 12 C57BL/6 mouse fetuses by
passage through a 26 gauge needle; debris was removed by gravity sedimentation
for 5 min on ice. Cell viability was determined by Trypan blue exclusion.
Progenitor cell enrichment was performed essentially as described (
24
) using Optilux 100 mm plastic Petri dishes (Falcon no. 1001; Becton Dickinson). Briefly, Petri dishes were coated with affinity-purified mouse anti-rat IgG (5 [mu]g/ml; Jackson Immunoresearch Laboratories, Jackson, ME) in 0.05 M Tris-HCl, pH 9.8, 0.15 M NaCl at 4oC overnight. After blocking with 3% fetal calf serum
(FCS)/balanced salt solution (BSS), 3 ml hybridoma supernatant, diluted 1:2,
was applied for 60 min. The rat antibodies were anti-AA4.1 (mAb AA4.1), anti-B220 (mAb 14.8), anti-Mac-1 (mAb M1/70) and anti Ly6A (E13 161). The dishes were
washed three times in 3% FCS/BSS and cell suspensions were applied to the
dishes and incubated at 4oC for 60 min. Non-adherent cells were removed by two washes in ice-cold 3% FCS/BSS. Adherent cells were recovered by scraping with
a plastic scraper (Costar no. 3010) after an additional eight washes.
Culture conditions and growth factors
Primary cell cultures were maintained in OPTI-MEM (Gibco BRL) supplemented with 10% FCS (Gibco BRL), 5 * 10
-5
M 2-mercaptoethanol (Sigma), 100 U/ml penicillin, 100 [mu]g/ml streptomycin (Gibco BRL) and the indicated growth factors. Murine MGF (Immunex Corp., Seattle, WA) was used at 100 ng/ml, IL-11 (Genetics Institute, Boston, MA) at 100 ng/ml and IL-7 (Immunex Corp, Seattle, WA.) at 100 U/ml.
Poly(A)
+
PCR
RESULTS
Identification of a cDNA fragment (7a3) by differential display
7a3 encodes a sialic acid-specific
O
-acetylesterase
Using the 155 bp cDNA fragment as a probe, a 70Z/3 cDNA library was screened,
resulting in the isolation of a 2.1 kb cDNA clone. The cDNA sequence was
submitted to nucleotide sequence databases (GenBank, EMBL, PDB and EST) and no
significant similarity to any recorded DNA sequences was found. However,
submission of the translated cDNA sequence to protein sequence databases (PIR,
SwissProt and GENPEPT) revealed similarity to a sialic acid-specific 9-
O
-acetylesterase from rat liver (PIR/A46690/B46690). This 9-
O
-acetylesterase, designated LSE, was found to consist of two disulfide
bonded subunits that arise from proteolytic cleavage of a single polypeptide chain (
15
). The predicted amino acid sequence encoded by 7a3 contains two regions of identity (88 and
83%), corresponding to the N-terminal sequences of both the small and large LSE subunits, respectively
(Fig.
2
). The similarity may actually be greater, since only the N-termini of the rat LSE protein subunits were sequenced and some residues
gave non-conclusive signals (Fig.
2
).
Analysis of the 7a3 sequence
Expression of 7a3 mRNA
Northern analysis of cell lines and tissues demonstrates that 7a3 mRNA is
expressed in cells of the B cell, T cell, myeloid and erythroid lineages, as
well as fibroblasts, stromal cell lines and non-hematopoietic tissues such as brain and liver (Fig.
3
). Analysis of B cell lines revealed that expression of 7a3 is developmentally regulated.
The 7a3 gene is expressed in the more mature B lineage cell lines 70Z/3, WEHI-231 and J558 and not in the less mature A-MuLV-transformed cell lines IIB4, CB17 1.1 and CB17 5.1 (Fig.
3
).
This pattern of expression prompted us to extend our studies to freshly isolated
primary fetal liver cells. We have previously shown that day 12 fetal liver
contains progenitors which give rise to B lymphocytes and macrophages (
29
). Three stromal cell-derived growth factors, IL-7, IL-11 and MGF, are sufficient to support the
in vitro
development of both committed B lymphocytes and macrophages from early
bipotential progenitors (
30
). We used this
in vitro
assay system to examine the expression pattern of 7a3 in developing B cells and
macrophages.
Expression of 7a3 was not detected in bipotential cells at the time of their
isolation, however, expression of 7a3 was detected as the cells differentiated. This was confirmed by three independent experiments, one of which is shown in Figure
4
. In these experiments, expression of 7a3 in cDNA samples made from 50 cells was examined (Fig.
4
and data not shown). Analysis of RNA from a total of 22 cell samples (50
cells/sample) failed to reveal expression of the 7a3 gene in bipotential cells
at the time of isolation or after 3 h culture. However, after 64 h culture 1/6
of the 50 cell samples and after 4 days 1/3 of the 50 cell samples expressed
the 7a3 gene.
DISCUSSION
A membrane-associated intralumenal sialate:9-
O
-acetylesterase (LSE) isolated from rat liver has been characterized biochemically (
15
). We have isolated a cDNA clone from mouse encoding a protein similar to the
rat LSE and have characterized the molecular structure. Structural features
include a 541 amino acid protein with a predicted hydrophobic leader sequence
and eight potential
N
-glycosylation sites. Six transcripts were revealed by Northern analysis
(Fig.
1
), suggesting that the 7a3 sequence may belong to a gene family or the
transcript may be extensively processed at the RNA level. Comparison of several
7a3 cDNA clones isolated from either screening a 70Z/3 cDNA library or
performing 5' RACE with 70Z/3 mRNA revealed a high degree of heterogeneity in the 5' sequences (Fig.
5
). Several observations indicate that the different 5' sequences probably arose from alternative splicing. First, in all clones these different 5' sequences join a common sequence at precisely the same residue. Second, the
consensus splice sequence extending into the 5' and 3' exons is present at each one of these junctions (
31
). Interestingly, the similarity to the N-terminus of the LSE small subunit begins immediately following the splice
junction site.
ACKNOWLEDGEMENTS
The authors are grateful to Dr Gillian Wu, Dr Stuart Berger, Dr Susan Zollman and Robert Ray for critical reading of the manuscript, to Dr Barbara Kee
for critical discussions and to Caren Furlonger for excellent technical
assistance. This work was supported by grants from the Medical Research Council
of Canada and the National Cancer Institute of Canada.
REFERENCES
1 Schauer,R. (1982) Sialic acids: Chemistry, Metabolism and Function, Cell Biology Monographs no. 10. Springer-Verlag, New York, NY.
17 Paige,C.J., Gisler,R.H., McKearn,J.P. and Iscove,N.N. (1984) Eur. J. Immunol., 14, 979-987.MEDLINE Abstract
18 Paige,C.J., Kincade,P.W. and Ralph,P. (1978) J. Immunol., 121, 641-647.MEDLINE Abstract
19 Bergman,Y.S., Stewart,S.J., Levy,S. and Levy,R. (1983) J. Immunol., 131, 1876-1881.
20 Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
21 Feinberg,A.P.V. and Vogelstein,B. (1983) Anal. Biochem., 132, 6-13.
