From the Department of Medicine and Clinical Science,
Kyoto University Graduate School of Medicine, 54 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan, ¶ Center for
Molecular Biology and Genetics, Kyoto University, 53 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606-8057, Japan, and the
Department of Nephrology, Saiseikai Nakatsu Hospital, Shibata
2-chome 10-39, Kita-ku, Osaka 530-0012, Japan
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ABSTRACT |
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Carbonic anhydrase (CA) is involved in various
physiological processes such as acid-base balance and transport of
carbon dioxide and ions. In this study, we have succeeded in the
isolation of a novel CA from the mouse kidney by use of the signal
sequence trap method. It is a 337-amino acid polypeptide with a
calculated molecular mass of 37.5 kDa, consisting of a putative
amino-terminal signal sequence, a CA domain, a transmembrane domain,
and a short hydrophilic carboxyl terminus, which we designated CA
XIV.1 The CA domain of
CA XIV is highly homologous with those of known CAs, especially
extracellular CAs including CA XII, IX, VI, and IV. The expression
study of an epitope-tagged protein has suggested that CA XIV is located
on the plasma membrane. When expressed in COS-7 cells, CA XIV exhibits
CA activity that is predominantly associated with the membrane
fraction. By Northern blot analysis, the gene expression of CA XIV is
most abundant in the kidney and heart, followed by the skeletal muscle,
brain, lung, and liver. In situ hybridization has revealed
that, in the kidney, the gene is expressed intensely in the proximal
convoluted tubule, which is the major segment for bicarbonate
reabsorption and also in the outer border of the inner stripe of the
outer medulla. In conclusion, we have cloned a functional cDNA
encoding a novel membrane-bound CA. This study will bring new insights
into our understanding of carbon dioxide metabolism and acid-base balance.
Carbonic anhydrase (CA)2
(EC 4.2.1.1) is a zinc-binding metalloproteinase that catalyzes
reversible hydration of carbon dioxide (CO2+H2O In the kidney, carbon dioxide metabolism and acid-base balance are
regulated by two CA isoenzymes, CA II and CA IV (2, 10). CA II
catalyzes hydration of carbon dioxide in the cytoplasm, and CA IV
catalyzes the reverse reaction on the plasma membrane. Thus, CA II and
CA IV, together, enhance the net proton secretion and bicarbonate
reabsorption (10). Functional importance of CAs has been investigated
also in pathologic conditions. For instance, CA II and CA IV are
up-regulated in the kidney in metabolic acidosis (11). Furthermore, CA
II gene mutations cause a hereditary disease characterized by renal
tubular acidosis, osteopetrosis, and cerebral calcification (12).
We have searched for novel soluble and membrane-bound proteins in the
mouse kidney using the signal sequence trap method (13-16). This
method enables isolation of not only signal transducing molecules such
as cytokines and receptors but also some sorts of enzymes and
transporters (13-19). Here we describe the molecular cloning, sequence
analysis, enzyme activity, and distribution of a novel membrane-bound
CA, designated CA XIV.
Tissue Preparation and RNA Extraction--
The whole kidney and
other tissues were obtained from 8-week-old male BALB/c mice. Total RNA
extraction was carried out as described by Chomczynski and Sacchi (20).
Poly(A)+ RNA was purified using PolyATract (Promega,
Madison, WI).
Signal Sequence Trap--
Signal sequence trap was performed as
described (13-16). 2 µg of poly(A)+ RNA from the mouse
kidney was reverse transcribed by random hexamer priming using
SuperScript II reverse transcriptase (Life Technologies, Inc.), and
deoxyadenosine (dA) tails were added at the 3' end of the first strand
cDNA. The second strand was synthesized with a specific primer
containing polydeoxythymidine (dT) and an EcoRI restriction
site and ligated with a SacI adaptor. The cDNA fragments of 300-700 base pairs in size were isolated by an agarose gel electrophoresis and subcloned into the EcoRI and
SacI sites of pcDL-SR Rapid Amplification of 3'-cDNA Ends (3'-RACE)--
The
3'-RACE experiment was performed using Marathon cDNA amplification
kit (CLONTECH Inc., Mountain View, CA) (24). 1 µg of poly(A)+ RNA from the mouse kidney was reverse transcribed by
oligo(dT)15 priming using SuperScript II reverse
transcriptase. After synthesis of the second-strand cDNA by the
method of Gubler and Hoffman (25) and ligation with the marathon
cDNA adaptor, polymerase chain reaction was carried out using a
5'-gene-specific primer (5'-TGGAAAATCAAGTCCCTGGAAGTC-3')
(nucleotides DNA Sequencing--
Nucleotide sequences were determined on both
strands by the dideoxy chain-termination method using Dye Terminator
cycle sequencing kit FS and 373B DNA sequencer (Applied Biosystems
Inc., Foster City, CA).
Phylogenetic Tree Construction--
Phylogenetic tree of CAs and
CA-related proteins was constructed by comparing the amino acid
sequences of their CA or CA-like domains using the alignment algorithm
of GeneWorks software (Oxford Molecular Group Inc., Campbell, CA),
which is based on the unweighted pair group method with arithmetic mean
(26). All parameters were set as default values. The proteins analyzed
were mouse CA XIV (amino acid positions 18-278), human CA I (2-261,
from GenBankTM accession number X05014), CA II (2-259,
Y00339), CA III (2-259, M29458), CA IV (19-285, M83670), CA V
(37-296, L19297), CA VI (19-278, M57892), CA VII (3-262, M76423), CA
IX (137-390, X66839), CA XII (28-289, AF037335/AF051882), RPTP Transient Expression and Measurement of CA Activity--
A
full-length mouse CA XIV cDNA (nucleotides Northern Blot Analysis--
Northern blot analysis was performed
as described with [32P]dCTP-labeled cDNA insert of
clone G31C5 (nucleotides In Situ Hybridization Analysis--
Sense and antisense
[35S]CTP-labeled cRNAs were generated from a partial
cDNA fragment of the 3'-RACE product (nucleotides Isolation and Sequence Analysis of Mouse CA XIV--
5,000 clones
from the mouse kidney cDNA library were screened by the signal
sequence trap method, and 25 positives were isolated. A positive clone
G31C5 with a 555-base pair insert encoded a novel 94-amino acid
polypeptide. To obtain the full-length cDNA, 3'-RACE was performed,
and a 1.6-kilobase fragment was obtained and sequenced. The full-length
cDNA encoded a novel CA-like protein with 337 amino acid residues,
which had a calculated molecular mass of 37.5 kDa (Fig.
1). Hydropathy analysis (32) predicted
that the protein consists of a signal sequence, a CA domain, a
transmembrane domain, and a short hydrophilic carboxyl terminus, which
we designated CA XIV. CA XIV possessed an N-glycosylation
motif in its CA domain and several potential phosphorylation sites by
protein kinases A and C in the carboxyl terminus (33). When clone G31C5
was expressed in COS-7 cells as an epitope-tagged protein, it was sorted to the cell surface and detected by cell surface immunostaining (see "Experimental Procedures"). These findings suggest that CA XIV
is located on the plasma membrane with its CA domain facing extracellular space.
The amino acid sequence of the CA domain of CA XIV was highly
homologous with those of other members of the CA family (Fig. 2). The amino acid identities were 43%
for human CA IX and XII, 38% for CA VI and VII, 35% for CA I, II,
III, and IV, 32% for CA V and VIII, and 31% for RPTP
Homology search using TBLASTN algorithm (35) revealed the
presence of partial cDNA fragments of the putative human
counterpart for CA XIV in the data bases, which are >80% identical to
mouse CA XIV at the amino acid level, e.g. expressed
sequence tag clones with GenBankTM accession numbers
H82563, R87427, and AA401879. The structural conservation beyond
species may suggest the physiological importance of CA XIV.
Phylogenetic Tree Construction--
To examine the evolutional
history of CA XIV among the CA family, a phylogenetic tree was
constructed (Fig. 2). The tree consisted of three clusters (3). The
first cluster included intracellular CAs (CA I-III, V, and VII), and
the third cluster included RPTP
As shown in this study, CA XIV likely localizes on the plasma membrane,
whereas CA IX is a membrane-bound protein that exists on the plasma
membrane and in the nucleus (6), CA VI is a secreted type of CA (4),
and CA IV is a CA attached to the plasma membrane by
glycosylphosphatidylinositol (GPI) linkage (36, 37). Thus, the second
cluster may be categorized as extracellular CAs. Consistent with their
subcellular localization, CA IV, VI, IX, XII, and XIV possessed
putative signal sequences in their amino termini (Fig. 2).
Measurement of CA Activity--
To examine whether CA XIV has CA
activity, the full-length cDNA was expressed in COS-7 cells. The
homogenate of mock-transfected cells exhibited low CA activity, which
may be attributed to endogenous CAs (Table
I). Transfection with the CA XIV cDNA
resulted in an increase in CA activity by 5.2-fold. The membrane
fraction of the homogenate possessed 2.1-fold higher activity than the whole homogenate. After the SDS treatment, which inactivates soluble CAs (29), the CA activity decreased only by 27%. These findings indicate that CA XIV is located predominantly in the membrane fraction.
Northern Blot Analysis--
Tissue distribution of CA XIV gene
expression was investigated by Northern blot analysis (Fig.
3). The CA XIV mRNA was 1.8 kilobases
in size and was expressed most abundantly in the kidney and heart,
followed by the skeletal muscle, liver, brain, and lung. No CA XIV
transcript was detected in the intestine and spleen. The tissue
distribution of CA XIV mRNA is very similar to that of CA IV
mRNA, which is expressed in the colon, lung, brain, kidney, and
heart but not in the intestine and spleen (37).
In Situ Hybridization Analysis--
The intrarenal localization of
CA XIV was determined by in situ hybridization analysis with
the antisense and sense cRNA probes (Fig.
4). At autoradiograph with the antisense
probe, strong hybridizing signals were observed in the cortex. At
photomicrograph, these signals were confined to the proximal convoluted
tubule, which is known as the most important segment for bicarbonate
reabsorption in the nephron (10). At autoradiograph, moderate signals
were also seen in the outer border of the inner stripe of the outer medulla. In view of shape, position, number, and comparison with previous reports using histochemical methods (38, 39), these signals
most likely correspond to the initial portion of long loop of the thin
descending limb of Henle. Physiological roles of CAs in the thin
descending limb of Henle still remain to be elucidated (10, 40).
Identification of specific inhibitors for CA XIV may facilitate our
understanding concerning such issues (2, 10). No specific signals were
seen in sections hybridized with the sense probe (not shown).
In the kidney, two CA isoenzymes, CA II and CA IV, have been well
characterized (10). CA II is a cytosolic isoenzyme localized in the
intercalated cells of the distal tubule and collecting duct, and the
thin descending limb of Henle (41, 42). On the other hand, CA IV is a
membrane-bound isoenzyme localized in the proximal convoluted tubule
and the thick ascending limb of Henle (43). Thus, the intrarenal
localization of CA XIV is distinct from those of CA II and CA IV,
suggesting its unique function in the kidney.
Comparison of CA XIV with CA IV--
CA XIV resembles CA IV in
that it has a putative signal sequence in its amino terminus, is
membrane-bound, and likely locates on the plasma membrane. Furthermore,
the tissue distribution of CA XIV is also very similar to that of CA
IV. However, CA XIV and CA IV differ in two points. First, the
intrarenal localizations of CA XIV and CA IV differ. Although they both
localize in the proximal convoluted tubule, CA XIV likely localizes
also in the thin descending limb of Henle, whereas CA IV localizes in
the thick ascending limb of Henle. Second, CA XIV is presumed to locate on the plasma membrane by a transmembrane domain, whereas, in case of
CA IV, the transmembrane domain in the extreme carboxyl terminus of the
precursor protein is cleaved off, and the mature protein is bound to
the plasma membrane by a GPI anchor (36, 37). The GPI-anchored form of
CA IV is released from the membrane by phosphatidylinositol-specific
phospholipase C (PI-PLC) treatment (36). Although CA IV was defined as
a membrane-bound isoenzyme of CA (2, 10), approximately half of the
membrane-bound CA activity in the kidney still remains after PI-PLC
treatment (29). One interpretation for this finding is that some part
of CA IV exists as a membrane-spanning form, which is PI-PLC
insensitive (29), yet it is also possible that other membrane-bound CAs are present in the kidney. A novel CA described here, CA XIV, is one of
such candidates.
Conclusion--
In this study, we describe the isolation and
characterization of a novel CA designated CA XIV. The primary structure
and the expression studies suggest that CA XIV is a type I membrane
protein localized on the plasma membrane. Because CA XIV mRNA is
expressed abundantly in various tissues and CA XIV has enzyme activity, CA XIV may play important roles in carbon dioxide metabolism and acid-base balance in the kidney and other tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
H++HCO3
).
CAs are produced in a variety of tissues where they participate in a broad range of physiological processes such as acid-base balance,
carbon dioxide, and ion transport, respiration, body fluid generation,
bone resorption, ureagenesis, gluconeogenesis, and lipogenesis (1, 2).
In mammals, CAs organize a family of 9 isoenzymes (CA I-VII, IX, and
XII) with defined CA activity, 3 CA-related proteins (CA VIII, X, and
XI), and 2 subtypes of receptor-type protein tyrosine phosphatase (RPTP
and
) (3). These proteins share a well conserved CA or CA-like
domain and differ in tissue distribution and subcellular localization
(1, 2). CA II is expressed in almost all tissues, whereas expression of
others are restricted (CA VI and CA VII in the salivary gland; RPTP
in the brain) (4, 5). CA IX and XII are expressed more abundantly in
various cancers than in corresponding normal tissues (6-8).
Subcellularly, they are cytosolic (CA I, II, III, and VII),
membrane-bound (CA IV, IX, and XII, RPTP
and
), mitochondrial (CA V), or secreted (CA VI). The CA-like domain of RPTP
does not
have CA activity, but provides a binding site for a cell surface signal
transducing molecule, contactin (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Tac(3') vector (21), to generate a
fusion protein with interleukin-2 receptor
chain (or Tac antigen)
lacking its own signal sequence (22). The expression plasmid library
thus obtained was transfected into COS-7 cells by the lipofection
method using Transfectam (Sepracor, Marlborough, MA). The fusion
proteins containing artificial amino-terminal signal sequences were
sorted to the cell surface, retained on the plasma membrane by a
transmembrane domain of Tac antigen, and detected by cell-surface
immunostaining with anti-Tac antibody (23). Otherwise, they remained
intracellularly and were not recognized by the antibody.
147 to
124, Fig. 1) and an adaptor primer
(5'-CCATCCTAATACGACTCACTATAGGGC-3'). The 3'-RACE product was subcloned
into the pCR II vector (Invitrogen Corp., San Diego, CA) for sequencing.
and
(34-300, M93426 and 56-321, L09247), and CA VIII (25-289, from
SwissProt accession number P35219).
12 to 1150) was
synthesized by reverse transcription-polymerase chain reaction using
the following primers: sense, 5'-TGTGGGGATAATATGTTGTTCTTCG-3'; antisense, 5'-GGGGTCCCTGGTGTATAGAGAGGG-3'. The nucleotide sequences were confirmed by sequencing. The cDNA was ligated into the
EcoRI restriction site of an expression vector pCXN2, a
derivative of pCAGGS (27). The plasmid was transfected into COS-7 cells
using Transfectam. Cells were harvested 72 h later and homogenized
in a buffer containing 50 mM Tris-SO4 (pH 7.5)
and 1 mM benzamidine (Sigma). The cell homogenate was
centrifuged at 100,000 × g for 30 min and separated
into the membrane fraction and cytosolic fraction. CA activity was
measured essentially as described by Sundaram et al. (28).
SDS-resistant CA activity was determined by measuring samples
preincubated with 0.2% SDS before assay (29). Protein concentrations
were determined by the Lowry method (30) using bovine serum albumin as
a standard. CA activity was calculated by the following formula: CA
activity (units/mg) = (T0/T
1)/C; where T0 represents the
reaction time for buffer alone (s); T, sample reaction time
(s); C, protein content (mg). The reaction times were
measured in triplicate, and the mean values were used to calculate CA activities.
265 to 282) (15). The blot was used to
expose BAS-III imaging plate (Fuji, Kanagawa, Japan) for 40 h.
147 to 698)
using T7 and SP6 RNA polymerases (Promega Corp.). In situ hybridization analysis was performed as described previously (15, 31).
In brief, 10-µm cryosections of the mouse kidney were mounted on
poly-L-lysine-coated slides, fixed with paraformaldehyde,
acetylated, and hybridized with the cRNA probes. Slides were washed,
dehydrated, and apposed to Hyperfilm
-max films (Amersham Int.,
Buckinghamshire, UK) for 10 days, or dipped into autoradiographic
emulsion (NTB-2, Eastman Kodak) and exposed for 10 weeks and
counterstained with hematoxylin and eosin.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequences
of CA XIV. Nucleotides and amino acids are numbered sequentially
from the translation initiation site. Box, putative
amino-terminal signal sequence and transmembrane domain; dotted
square, region obtained by signal sequence trap;
underline, in-frame termination codon preceding the
initiation codon; , termination codon; #, putative zinc-binding
histidine residues;
, putative CA active site residues;
,
potential N-glycosylation site; square, potential
phosphorylation sites by protein kinase A (
) and protein kinase C
(
).
and
(3).
The homologies were especially high in the putative CA active site
residues including 3 conserved zinc-binding histidine residues (Fig. 1)
(3, 34). When CA XIV was compared with CA VII, which is considered the common ancestor of the CA family (3), as many as 30 of 36 active site
residues were identical. These findings imply that CA XIV possesses CA
activity (3).
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Fig. 2.
Phylogenetic tree, domain scheme, and
subcellular localization of CAs and CA-related proteins.
Phylogenetic tree was constructed by comparing the amino acid sequences
of the CA or CA-like domains using an alignment algorithm based on the
unweighted pair group method with arithmetic mean (26). Values indicate
estimated genetic distances, which are proportional to the lengths of
horizontal lines. Thick lines at branching points represent
error bars whose lengths indicate standard errors of the branch
positions. Note that all proteins are from human, except CA XIV from
mouse. CA VIII is also called CA-related protein; CA IX, MN protein;
RPTP , receptor-like protein tyrosine phosphatase
.
, putative
signal sequence; CA, CA or CA-like domain; TM, transmembrane domain;
GPI, anchoring site to glycosylphosphatidylinositol; PTPase, protein
tyrosine phosphatase domain.
and
. CA XIV was most closely
related to CA XII, followed by CA IX, VI, and IV, forming the second cluster.
CA activity of CA XIV cDNA-expressing COS-7 cells
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Fig. 3.
Tissue distribution of CA XIV gene
expression. Northern blot was performed by 32P-labeled
cDNA probe with 50 µg of total RNA loaded in each lane. Positions
of 28 S and 18 S ribosomal RNAs are indicated on the left. 28 S
ribosomal RNA bands visualized by ethidium bromide are shown at the
bottom.
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Fig. 4.
Intrarenal localization of CA XIV gene
expression. In situ hybridization was performed by
35S-labeled cRNA probe. A, autoradiograph with
the antisense probe (magnification, ×10). B, bright-field
photomicrograph of cortex (magnification, ×150). Cx,
cortex; OM, outer medulla; IM, inner medulla;
OS, outer stripe; IS, inner stripe; G,
glomerulus.
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ACKNOWLEDGEMENTS |
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We thank Prof. T. Honjo, Department of Medical Chemistry, Kyoto University Graduate School of Medicine and Prof. T. Nakano, Research Institute for Microbial Diseases, Osaka University for encouragement, and Prof. J. Miyazaki, Department of Nutrition and Physiological Chemistry, Osaka University Medical School for kindly providing an expression vector pCXN2. We also acknowledge Dr. T. Nakamura, Department of Medical Chemistry, Kyoto University Graduate School of Medicine for discussions and T. Aoki, S. Koide, and E. Nakano for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by research grants from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists, the Japanese Ministry of Education, Science, Sports, and Culture, and the Japanese Ministry of Health and Welfare.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AB005450.
§ To whom correspondence should be addressed: Tel.: 81-75-751-3173; Fax: 81-75-771-9452; E-mail: ogawa{at}kuhp.kyoto-u.ac.jp.
1 The designation CA XIV for the cloned protein and Car14 for the mouse gene has been approved by the Specialist Advisor for Carbonic Anhydrases with the Human Gene Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature).
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ABBREVIATIONS |
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The abbreviations used are: CA, carbonic anhydrase; RPTP, receptor-type protein tyrosine phosphatase; 3'-RACE, rapid amplification of 3'-cDNA ends; GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C.
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