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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 340-346
TRANSFUSION MEDICINE
Expression of Kell blood group protein in nonerythroid tissues
David Russo,
Xu Wu,
Colvin M. Redman, and
Soohee Lee
From the Lindsley F. Kimball Research Institute, The New York Blood
Center, New York, New York.
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Abstract |
The Kell blood group protein is a zinc endopeptidase that yields
endothelin-3, a potent bioactive peptide, by cleavage of big
endothelin-3, a larger intermediate precursor. On red cells, Kell
protein is linked by a single disulfide bond to XK, a protein that
traverses the membrane 10 times and whose absence, as occurs in the
McLeod phenotype, is associated with a set of clinical symptoms that
include nerve and muscle disorders and red cell acanthocytosis.
Previous studies indicated that Kell is primarily expressed in
erythroid tissues, whereas XK has a wider tissue distribution. The
tissue distribution of Kell protein has been further investigated by
Northern blot analysis, PCR-screening of tissue complementary DNAs
(cDNAs), and Western immunoblots. Screening of an RNA dot-blot
panel confirmed that Kell is primarily expressed in erythroid tissues
but is also expressed in a near equal amount in testis, with weaker
expression in a large number of other tissues. PCR-screening of cDNAs
from different tissues and DNA sequencing of the products gave similar
results. In 2 of the nonerythroid tissues tested, testis and skeletal
muscle, Kell protein was detected by Western immunoblotting. In
skeletal muscle, isolation of XK with a specific antibody coisolated
Kell protein. These studies demonstrate that Kell is expressed in both erythroid and nonerythroid tissues and is associated with XK.
(Blood. 2000;96:340-346)
© 2000 by The American Society of Hematology.
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Introduction |
Kell is a polymorphic 93 kDa type II membrane
glycoprotein1 that on human red cells carries the majority
of the Kell blood group antigens.2,3 Kell protein is a
member of the neprilysin (M13) family of zinc endopeptidases, and it
preferentially cleaves big endothelin-3 at a Trp21-Ile22 bond, yielding
the 21-amino acid bioactive peptide, endothelin-3.4
Previous Northern blot studies indicated that Kell is expressed
primarily in bone marrow and fetal liver, with little or no Kell
transcripts noted in brain, kidney, adult liver, and lung. In the
erythroid tissues, the major Kell transcript was 2.5 kilobase (kb), but
smaller amounts of larger transcripts, 6.6, 11.5, and 13.2 kb, were
also observed.5
Early serological studies had predicted an association of Kell with
another red cell membrane protein, XK, which expresses a surface
antigen, termed Kx.2 XK is a 444-amino acid protein that is
predicted to traverse the membrane 10 times.6 Although the
function of XK is not known, its absence from red cells, the McLeod
phenotype, is associated with a set of clinical symptoms that include
red cell acanthocytosis, elevated levels of serum creatine
phosphokinase, and late onset forms of muscular dystrophy and nerve
abnormalities, characterized by areflexia and chorea. A covalent
linkage between Kell and XK was shown, in that Kell and XK were
coisolated by immunoprecipitation in nonreduced
conditions.7,8 Site-directed mutagenesis of specific
cysteine residues in Kell and XK proteins and coexpression in
transfected cells demonstrated that Kell Cys72 is disulfide-linked to
XK Cys347.9 Because XK had been shown to be expressed not
only in erythroid tissues but also in skeletal muscle, pancreas, heart,
brain, placenta, and several other tissues,6 we have
reinvestigated the tissue distribution of Kell with the use of Northern
blot analysis, PCR-screening of human tissue complementary cDNAs
(cDNAs), and Western immunoblots. We find that Kell is also expressed
in many nonerythroid tissues, primarily in testis, and that in the
nonerythroid tissues, as exemplified by skeletal muscle, Kell is
disulfide-linked to XK.
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Materials and methods |
RNA dot-blot analysis
A dot-blot panel, containing 89-514 ng of 50 different human tissue
poly A+ RNAs, normalized for equal amounts of actin
messenger RNA (mRNA), was purchased from Clontech (Palo Alto, CA). The
panel, which also contained several RNA and DNA controls, was
hybridized to a 32P-labeled 0.44-kb antisense RNA covering
KEL exons 9 to 13.10 The riboprobe was synthesized
by SP6-directed reverse transcription of a fragment of the Kell cDNA,
using the Strip-EZ RNA kit (Ambion, Austin, TX).
Northern blot analysis
Poly A+ RNA from various tissues, separated by
electrophoresis and transferred to membranes, was purchased from
Clontech. The membranes were hybridized at 68°C with
32P-labeled Kell antisense mRNA. The riboprobe was
synthesized by T7-directed reverse transcription of 2.4-kb Kell
cDNA,1,10 using the Strip-EZ RNA kit purchased from Ambion.
A random primed actin cDNA probe was synthesized, using the Strip-EZ
DNA kit (Ambion).
PCR screening of KEL in human tissue cDNAs
First, strand cDNA preparations, normalized for equal amounts of 6 different housekeeping cDNAs, from 20 different human tissues were
purchased from Clontech. About 1 ng of cDNA from each of the different
tissues was used as a template for 25 cycles of PCR amplification as
recommended by the manufacturer. The following primers, derived from
exons 14 and 18 of the KEL gene,10 were used:
5'-TTG CAG CCT CAC CCC CAA CAC AGG TGG A-3' and
5'-AGC CCC CCA ACG TCT GCA GCA TTC TCT A-3'.
The cDNA preparations were also used for detection of glycophorin A
(GPA), using the following primers derived from the GPA gene11: 5'-CAG ACA AAT GAT ACG CAC AAA CGG
GAC-3' and 5'-CTT TAT CAG TCG GCG AAT ACC GTA AGA-3'.
One of the primers contained sequences spanning exons 2 and 3, and the
other was derived from exon 5.
PCR amplification of a skeletal muscle cDNA containing the Kell
coding region
A Kell cDNA from a muscle cDNA library (Human Skeletal Muscle,
Marathon-Ready, cDNA library, Clontech) was PCR-amplified by the nested
3' end RACE procedure.
The following forward primers, specific for Kell sequences, were used:
5'-ATG GAA GGT GGG GAC CAA AGT GAG GAA G-3' and
5'-GCC GAG GGA ACG CAG CCA GGC AGG TGG A-3'.
The first primer, nt 121-149, which includes the initiation codon in
KEL exon 1, was used with adapter primer 1 (AP1), present at
the 3' end, in a first round of PCR amplification. The second primer, nt 150-177, from KEL exon 2, was used with adapter
primer 2 (AP2), which is internal to AP1. A 2.4-kb PCR product was
obtained, covering from nt 150 to the 3' end of Kell
cDNA.1,10
The 5' transcription start site was obtained by 5' RACE
nested polymerase chain reaction (PCR), using the following
KEL-specific reverse primers: 5'-CTC CTC CCC AGA GCC TGG
GTG CCA GGA ATT-3' and 5'-CTG GCC ACT GCC CAT GGC CTG CTC
CCT TCC-3'.
The first primer, derived from KEL exon 5, was used with AP1
present at the 5' end, and the second primer, representing
sequences from KEL exon 3, was used with AP2 that is internal
to AP1. The PCR-amplified products were sequenced, using AP2 and the
primer from exon 2.
DNA sequencing of PCR products
PCR-amplified products were separated by agarose gel
electrophoresis, the bands excised and eluted for direct sequencing, using the Genclean kit, glassmilk-based method (Bio 101, Vista, CA).
The PCR-amplified fragments from the various tissue cDNAs were
sequenced, using the amplification primers.
The following Kell primers were used for sequencing the 2.4-kb
PCR-derived Kell skeletal muscle cDNA: 5'-GCC GAG GGA ACG CAG CCA
GGC AGG TGG A-3' derived from KEL exon 2; 5'-GGT
CCA GAA TTC CTG GCA CCC AGG C-3' from exon 5 and 5'-CTC CTC
CCC AGA GCC TGG GTG CCA GGA ATT-3' from exon 5; 5'-GCT TGC
CCT GTG CCC GCC GCT GCT C-3' from exon 8; 5'-TGG CTG AGC
TTT CTG CGT GCC TCC TGG A-3' from exon 10 and 5'-GGA CTT
TCT GCA GAG CCA CAT GAT C-3' from exon 10; 5'-GGG TTG GAG
GAG TCC AGC TGG AAA G-3' from exon 15; 5'-ATG GCC CAC GAG
CTG TTG CA-3' from exon 16; 5'-AGC CCC CCA ACG TCT GCA GCA
TTC TCT A-3' from exons 17-18; and 5'-GGT GTT GGT CGA TAT
TTC TGT GCT GTG GC-3' from exon 19.
The Microchemistry Laboratory of the New York Blood Center performed
DNA sequencing with the use of an ABI 373XL sequencer (Perkin-Elmer,
Applied Biosystems, Foster City, CA), procedures from the manual, and
ABI Big Dye reagents with BD Half-Term (Gen Pak).
Antibodies
Four antibodies were used in this study. (1) An antibody to the
second extracellular loop of XK was used in immunoprecipitation studies, as previously described.9 This antibody was tagged with biotin, using sulfosuccinimidyl 6-(biotinoamido) hexanoate (sulfo-NHS-LC-biotin; Pierce, Rockford, IL), according to the manufacturer's directions. (2) Kell protein, isolated from red blood
cells, was used to generate a polyclonal antibody in rabbits. (3) An
antibody to the C-terminal intracellular domain of XK was raised in
rabbits by treatment with a 39 mer synthetic peptide, corresponding to
XK amino acids 406 to 444. The Microchemistry Laboratory of the New
York Blood Center prepared the synthetic peptide. This peptide was also
used to affinity purify the antibody. By Western blot assay, this
antibody reacted with XK and XK/Kell complex from red cells of common
Kell phenotype but not with proteins of the same size from McLeod red
cells. The antibody did, however, have some cross-reaction with band 3 proteins from both McLeod and normal red cells. (4) A monoclonal
antibody to glycophorin A and B was purchased from Sigma (St
Louis, MO).
Preparation of an integral membrane fraction
Treatment of cell membranes with sodium carbonate at alkaline pH
removes peripheral proteins and retains integral membrane proteins.12 This procedure was used to prepare cell
membranes from various tissues. Frozen human tissues (testis and
skeletal muscle) were obtained from the National Disease Research
Interchange (NDRI; Philadelphia, PA). While still frozen
( 70°C), about 2 grams of tissue was minced using a nickel
scalpel, placed in 20 volumes of ice-cold 100 mmol/L sodium carbonate
buffer, pH 11.5, and homogenized using a glass tissue grinder. The
suspension was filtered through cheesecloth, and centrifuged at 4000 rpm for 10 minutes at 4°C in a Sorvall model RC-5C plus to remove
unbroken cells and nuclei. The supernatant fractions were then
centrifuged at 40 000 rpm for 30 minutes at 4°C with the use of a
Beckman 60Ti rotor, and the pellet, composed mostly of cellular
membranes, was rinsed twice with phosphate-buffered saline (PBS). The
membranes were then solubilized by homogenization in 5 volumes of lysis buffer composed of PBS, containing 1% n-dodecyl  -d-Maltoside (Sigma), 0.5% of deoxycholic acid (sodium salt, Sigma), and protease inhibitors. The following protease inhibitors were used: 0.1 mmol/L L-1-tosylamido-2-phenylethyl chloromethyl ketone, 0.1 mmol/L
phenylmethylsulfonyl fluoride, and 10 units/mL aprotinin (Sigma). The
solution was than cleared by centrifugation in a Beckmann 50Ti rotor at
27 000 rpm for 20 minutes.
Isolation of XK complexes from nonerythroid tissues
The detergent-solubilized membrane preparation, described above, was
incubated with biotinylated antibody to XK at 4°C overnight. The
immunocomplex was recovered by adding 0.02 volumes of immobilized streptavidin suspension (Pierce), at 4°C for 3 hours with constant rocking. The streptavidin gel was then packed in a 5-mL column and
washed 4 times with lysis buffer, and the XK complex was eluted with
100 mmol/L glycine buffer pH 2.8. The eluted material was collected in
tubes containing 1 mol/L Tris to neutralize acidity.
SDS-PAGE and Western blotting
Membrane preparations, or isolated XK complexes, were dissolved in
SDS-buffer (0.125 mol/L Tris-HCl, pH 6.8, 1% SDS, 5% glycerol), separated on precasted 4%-12% Tris-glycine gradient gels (Novex, San
Diego, CA) and transferred to nitrocellulose membranes (Schleicher and
Schuell, Keene, NH) with the use of a Bio Rad (Hercules, CA) blotting
apparatus. After treatment with rabbit antibodies, 2 different
detection procedures were employed. The second antibody was conjugated
either with alkaline phosphatase or horseradish peroxidase. Both second
antibodies were purchased from Pierce. The alkaline
phosphatase-conjugated antibody was detected with a chromogenic
substrate (NBT/BCIP; purchased from BIORAD) and the horseradish
peroxidase-conjugated antibody with a chemoluminescent substrate
(Supersignal; purchased from Pierce).
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Results |
Detection of Kell RNA transcripts in nonerythroid tissues
Hybridization of a RNA dot-blot panel with radioactive Kell
antisense RNA followed by densitometry confirmed that Kell is expressed
mainly in fetal liver and bone marrow (Figure
1; Table 1).
However, significant hybridization also occurred in many other tissues
with the highest in testis. Weaker signals were observed throughout the
dot-blot panel, including different parts of the brain and many other
tissues. No hybridization occurred in the controls that contained
nonhuman RNA or human DNA (Figure 1).
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| Fig 1.
Dot-blot analysis of different tissues.
(Top) An autoradiography of a dot blot containing 51 different human
tissue RNAs hybridized with a Kell radioactive riboprobe. (Bottom)
Relative concentration values obtained by densitometry analysis. Tissue
RNAs can be identified according to their position on the blot and
their number as summarized in Table 1.
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Northern blot analysis of 2 panels containing a total of 15 different
human tissue RNAs showed that the 2.5-kb and 6.6-kb Kell transcripts
present in bone marrow (Figure 2, lane 1)
were also abundant in testis (Figure 2, lane 12). The same size
transcripts were also detected in lesser amounts in lymph node, colon,
and spleen (lanes 4, 9, and 15). Weaker signals for the 2.5-kb
transcripts were detectable in several other tissues (Figure 2),
including skeletal muscle (data not shown). Because of different
amounts of mRNA, as evidenced by the actin signals, the relative
amounts of Kell transcripts cannot be easily compared. Some tissues,
such as small intestine (Figure 2, lane 10), had less actin mRNA than the other tissues. Other tissues, such as thyroid and peripheral blood
lymphocytes (PBLs), showed transcripts of a different size than
normally obtained for Kell transcripts. Thyroid had a smaller transcript of about 1.3 kb, and PBLs had a band at 3.5 kb. On occasion
(as shown in Figure 2, lane 1), bone marrow also has a small,
approximately 1 kb, transcript that may be caused by mRNA degradation.
However, relative values were obtained by comparing the densities of
the main 2.5-kb transcripts and correcting them for the different
amounts of actin (Figure 1). The results presented in Table
2 show that, unlike the results in Figure
1, KEL appears to be expressed more in testis than in bone
marrow.
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| Fig 2.
Northern blot analysis.
Lanes 1 to 15 contain approximately 2 µg poly A+ RNA from
(in the following order): bone marrow, adrenal gland, trachea, lymph
node, spinal cord, thyroid, stomach, peripheral blood leukocytes,
colon, small intestine, uterus, testis, prostate, thymus, and spleen.
The 2.5-kb and 6.6-kb Kell transcripts are marked by arrows.
Hybridization was performed with radioactive Kell antisense RNA (upper
panel) or with radioactive  -actin cDNA (lower panel).
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PCR analysis of human tissue first strand cDNAs, followed by DNA
sequencing of the amplified DNA fragments, was conducted to confirm the
presence of the Kell transcript in various non-erythroid tissues. A
total of 20 tissue cDNAs of erythroid and nonerythroid origins were
analyzed for the presence of Kell. Primers for PCR were designed to
anneal to sequences derived from exon 14 and exon 18 of the KEL
gene to amplify a 386-base pair (bp) DNA fragment. A PCR product with
the expected size was detected in all tissues, with the highest amounts
in bone marrow, fetal liver, tonsils, spleen, placenta, and testis
(Figure 3, lanes 3, 5, 6, 7, 15, and 21).
No PCR product was detected in the negative control (Figure 3, lane 1),
which had water instead of cDNA template. DNA sequencing analysis of
the 386-bp DNA fragment from 4 of the samples, testis, adult liver,
PBLs, and skeletal muscle, confirmed that the PCR-amplified products
had identical sequences to that obtained from bone marrow or fetal
liver. These tissues were chosen because there is abundant expression
of Kell in testis, because there is very weak expression in skeletal
muscle, and because previous studies had not detected Kell transcripts
in adult liver and PBLs.
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| Fig 3.
PCR analysis of Kell cDNA.
Normalized first strand cDNA preparations from different human tissues
as indicated below were screened by PCR. Primers were designed to
amplify a 386-bp segment (arrow). (1) Control (no template cDNA), (2)
peripheral blood leukocytes, (3) bone marrow, (4) lymph node, (5) fetal
liver, (6) tonsils, (7) spleen, (8) thymus, (9) brain, (10)
heart, (11) kidney, (12) liver, (13) lung, (14) pancreas,
(15) placenta, (16) skeletal muscle, (17) colon, (18) ovary, (19)
prostate, (20) small intestine, and (21) testis.
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The complete DNA sequence of Kell cDNA obtained from skeletal muscle
was further investigated by 3' and 5' RACE nested PCR and
DNA sequencing. A 3' RACE DNA fragment, of about 2.4 kb, was generated, using primers derived from KEL exon 1 and adapter
primers at the 3' end, thus containing the whole coding region.
DNA sequence analysis showed that the coding region of the skeletal
muscle Kell cDNA was nearly identical to that previously described from bone marrow Kell cDNA, with only one difference: a G to A mutation at
nt 2197. This base change would encode an asparagine at Kell residue
692 instead of aspartic acid.
The 5' untranslated region of skeletal muscle cDNA was obtained
by 5' RACE, sequenced, and found to be identical to
Kell fetal liver and bone marrow cDNA.
Because the tissues could be contaminated with erythroid cells during
isolation, and because PCR is a sensitive technique capable of
amplifying small amounts of cDNA, we evaluated this possibility with
the use of primers specific for GPA, which is expressed only in
erythroid tissues. If blood cells contaminated the non-erythroid
tissues, GPA should be amplified, as well as Kell. Nine different
tissue cDNAs, including bone marrow and fetal liver, were analyzed for
GPA, using the same PCR conditions as for amplification of Kell
transcripts. In bone marrow and fetal liver, primers derived from
sequences of exons 2-3 and exon 5 of the GPA gene amplified a DNA
fragment with the expected size of 330 bp (Figure
4, lanes 9 and 10). By contrast, no GPA
products were observed in any of the nonerythroid tissues (Figure 4,
lanes 2 to 7), thus showing that erythroid contamination was not
relevant and could not account for the amplification of Kell cDNA in
non-erythroid cDNA libraries.
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| Fig 4.
PCR analysis of GPA cDNA.
Normalized first strand cDNA preparations from different human tissues
as indicated below. Primers were designed to amplify a 330-bp segment
(arrow). (1) Control (no template cDNA), (2) brain, (3) skeletal
muscle, (4) lung, (5) liver, (6) spleen, (7) lymph node, (8) peripheral
blood leukocytes, (9) bone marrow, and (10) fetal liver.
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Kell protein is present in testis and skeletal muscle
The presence of Kell protein in nonerythroid tissues was determined
by direct Western immunoblotting of integral membrane proteins in
testis and skeletal muscle. Membrane preparations, containing integral
membrane proteins, were prepared by treatment of the membranes with
Na2CO3 at pH 11.5. The proteins were separated by SDS-PAGE in reducing conditions and detected by Western
immunoblotting, using a polyclonal antibody to Kell protein isolated
from red cells. Kell protein, having the same electrophoretic mobility as that from red cell membranes, was detected in both testis and skeletal muscle (Figure 5, left panel).
Unequal amounts of integral membrane proteins were loaded for the
different tissues. The lanes containing skeletal muscle had
about 35 µg protein, testis 15 µg protein, and red cells
5 µg protein.
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| Fig 5.
Western blot analysis.
Integral membrane proteins (pH 11.5) from red blood cells, testis, or
skeletal muscle were reduced, separated by SDS-PAGE, blotted, and
detected with polyclonal antibody to Kell (left panel) or with a
monoclonal antibody that recognizes glycophorins A and B (right panel).
(Left panel) A single 93-kD band (arrow), the molecular size of Kell,
was detected in red blood cells, testis, and skeletal muscle. (Right
panel) Multiple bands corresponding to glycophorins A and B were
detected in red blood cells but not in testis or skeletal muscle.
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A duplicate Western immunoblot, using monoclonal antibody to both
glycophorin A and B, was performed to evaluate the possible contamination of testis and skeletal muscle with red cells. The antibody to the glycophorins reacted with multiple red cell membrane proteins but did not react with proteins from testis or skeletal muscle
(Figure 5, right panel).
Association of Kell and XK in nonerythroid tissues
Previous studies7-9 demonstrated that Kell and XK are
covalently linked on the red cell membrane. To determine whether Kell is also bound to XK in nonerythroid tissues, XK and its complexes were
isolated from skeletal muscle membranes, using a biotinylated antibody
to XK and characterized by Western immunoblotting, with the use of
antibodies to Kell or to XK. Under reducing conditions, the antibody to
the C-terminal domain of XK reacted with XK and with a higher molecular
weight protein with the expected size of XK dimer (Figure
6, second lane right panel). In
reducing conditions, the presence of Kell protein was shown
by reaction of antibody to Kell with a 93-kDa protein (Figure
6, first lane left panel). Under nonreducing conditions, both
antibodies recognized the Kell/XK protein complex, with a molecular
weight of about 130 kDa (Figure 6, lane 2 left panel and lane 1 right
panel), demonstrating the presence of a Kell/XK complex in skeletal
muscle.
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| Fig 6.
Kell/XK complex in skeletal muscle.
Skeletal muscle integral membrane proteins were isolated with antibody
to XK, separated in reduced and nonreduced conditions by SDS-PAGE, and
detected with antibody to Kell (left panel) or to XK (right panel). The
location of Kell/XK complex, Kell, and XK are marked by arrows.
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Discussion |
Our studies show that expression of Kell protein, like that of its
partner, XK, is not limited to erythroid tissues but is widely
distributed. This finding is in keeping with the previous observation that Kell and XK are covalently linked on red
cells.7-9 Earlier studies, in which expression of Kell and
XK were determined only by Northern blot analysis, did not find that
these 2 proteins were expressed in the same tissues, because expression
of Kell was noted only in erythroid tissues and not in brain, adult
liver, kidney, and lung,5 whereas expression of XK was
detected in erythroid and nonerythroid tissues, primarily skeletal
muscle, pancreas, heart, and brain.6 Our studies now
indicate that Kell, like XK, is also expressed in nonerythroid tissues,
primarily in testis, but with significant amounts in other tissues.
Kell expression was determined by 2 different Northern blot assays. One
of the procedures measured a large number of tissues as part of a
dot-blot panel in which the amount of poly A+ RNA in each
well was varied to reflect equal amounts of actin poly A+
RNA. The Kell probe used was a riboprobe covering the middle segment of
Kell mRNA. Although this segment of Kell, representing exons 9 to 13 of
KEL, is not highly homologous with other members of the M13
family of zinc endopeptidases,1 there could be
cross-reaction with other M13 RNAs. Because the other M13
members have larger mRNA transcripts,13-19 Northern
blot analysis, detecting the major 2.5-kb and 6.6-kb Kell transcripts,
was also performed. In general, the 2 procedures matched, with abundant
Kell expression detected in erythroid tissues and testis and with
weaker expression in other tissues. However, not all of the
tissues represented in the dot-blot panel were tested by Northern
blot analysis for the presence of 2.5-kb and 6.6-kb transcripts, and
lengthy exposures of the film were necessary to detect Kell transcripts
in tissues other than testis and the erythroid tissues. In the dot-blot
experiments (Figure 1), there was near-equal expression of KEL
in both bone marrow and testis, whereas, by Northern blot analysis
(Table 2), there was twice as much expressed in testis. The differences
in the 2 procedures may be due to the fact that in the dot-blot assays total KEL transcripts were measured, whereas in the Northern
blots only the main 2.5 kb was taken into account. Thus, any
degradation of RNA that would lead to smaller transcripts, for example
the 1-kb transcript in bone marrow (Figure 2), is not measured and will
contribute to a lower value. Nevertheless, all procedures indicate that
erythroid tissues and testis are the major sites of Kell expression.
PCR screening of cDNA from many tissues also supports that Kell is
widely distributed. The 2 primers used to amplify the Kell transcripts
are in areas that are homologous with sequences for NEP and ECE-1, and
some amplification of these transcripts may have occurred. Sequencing
of several of the transcripts showed, however, that only Kell
transcripts were amplified. The PCR procedure employed is not
quantitative, although it does give a rough indication of the relative
distribution of Kell transcripts. The PCR method is limited by
variations in the preparation of poly A+ RNA from different
tissues and by the fact that the PCR procedure used is not
quantitative. Thus, some minor discrepancies are found between the
Northern blot analysis and the PCR determinations. For example, Kell
appears to be moderately expressed in colon by Northern blot analysis
(Figure 2, lane 9), but very little was detected by PCR screening
(Figure 3, lane 17).
Because Kell is abundantly expressed in erythroid tissues, and the
presence of erythroid cells may easily contaminate human tissues, it
was important to rule out the possibility that the detection of Kell
was due to contamination of tissues with blood. GPA is much more
abundant in erythroid tissues than Kell, but PCR screening did not
detect GPA in nonerythroid tissues, indicating that contamination with
erythroid tissues is not a concern.
The presence of mRNA, as measured by Northern blot or RT-PCR, may not
accurately reflect the presence of protein because other cellular
mechanisms, such as protein stability or turnover, can affect protein
content. Therefore, we determined, by Western immunoblot, the presence
of Kell protein in 2 different tissues. We chose testis because both
Northern blot and PCR procedures indicate that Kell is abundantly
expressed in this tissue. For contrast, we also examined skeletal
muscle that abundantly expresses XK as determined by Northern blot but
shows weak expression of Kell by Northern blot and PCR (Figures 1 and
3). Kell protein was present in both testis and skeletal muscle.
According to different amounts of total integral membrane proteins
loaded on the gels, we estimate that testis has twice as much Kell
protein as skeletal muscle and that red cell membranes may have 7 times
more than skeletal muscle. In skeletal muscle, most of XK isolated was
complexed to Kell and very little appeared as free XK. However, this
procedure may not account for all of the free XK protein because we
have noticed that the antibody to XK preferentially reacts with XK when
it is complexed to Kell and that free XK is more difficult to detect.
The Kell/XK complex may be more stable than the individual component
proteins. This view is based on studies with 2 rare Kell red cell
phenotypes in which either Kell or XK is absent or
deficient.2 In the McLeod red cell phenotype, which lacks
or is deficient in XK, there is also diminished amounts of Kell
protein; whereas in the Ko (null) phenotype, which lacks Kell protein,
there is less XK protein on the membrane, although an antigen Kx,
carried by XK, appears to be enhanced.8 Other studies have
shown that cellular transport and placement of Kell and XK on the
plasma membrane does not require coexpression of both Kell and XK. In transfected COS cells, expression of Kell or XK by themselves is
sufficient to allow the individual proteins to be expressed on the cell
surface.20 In normal adult red cells, however, there is
little free Kell or XK, and the majority of these proteins exists as a
130-kDa Kell/XK complex.
We do not know, however, whether the Kell/XK complex in nonerythroid
tissues is similar to that in red cells. Red cells have a single
membrane representing the plasma membrane and, by contrast, nonerythroid tissues may have multiple intracellular membranes. The
enzymatic activity of Kell protein is optimal at an acidic pH,4 suggesting that Kell may play an intracellular role,
possibly in transport and processing of endothelin precursors or in the endocytic pathway. In this regard, it should be noted that
endothelin-converting enzymes are not only ectoenzymes but are also
present intracellularly.21-24 Future immuno-histochemical
studies are necessary to determine the cellular and intracellular
locations of Kell and XK. Whether the full cellular function of Kell
requires XK, or whether Kell is complementary to the function of XK,
also remains to be determined. Thus far, big endothelin-3 is the
preferred substrate for Kell, as determined by in vitro studies with
recombinant Kell and by measuring enzymatic activity of red
cells.4 We do not know, however, if big endothelin-3 is the
only substrate for Kell and whether Kell, which may be present
intracellularly, has a different function to Kell present on cell surfaces.
DNA sequencing of a Kell cDNA obtained by PCR amplification from a
skeletal muscle cDNA library showed that DNA sequence was identical to
that obtained from erythroid tissues with the single exception of a
possible Asp 692 Asn substitution because of a single base change. This
single base change may, however, be due to the PCR procedure and will
have to be confirmed by other methods. The sequence identity between
erythroid and non-erythroid Kell suggests similar enzymatic functions
in erythroid and nonerythroid tissues. The fact that Kell is
disulfide-linked to XK in nonerythroid tissues also indicates
complementary functions for XK and Kell proteins.
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Acknowledgments |
We thank Dr James D. Farmar and Andrea Molinaro of the Laboratory of
Microchemistry for their work in DNA sequencing.
| |
Footnotes |
Submitted September 20, 1999; accepted February 28, 2000.
Supported in part by a National Institutes of Health
Specialized Center of Research (SCOR) grant in Transfusion Biology and Medicine, HL54459.
Reprints: Colvin M. Redman, The New York Blood Center,
310 East 67 St, New York, NY 10021; e-mail credman{at}nybc.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction