Research Article Changes in red cell ion transport, reduced intratumoral neovascularization, and some mild motor function

abnormalities accompany targeted disruption of the Mouse Kell gene (Kel)
Xiang Zhu,1 Alicia Rivera,2 Mari S. Golub,3 Jianbin Peng,1 Quan Sha,1 Xu Wu,1 Xiaoling Song,1 Prem Kumarathasan,4 Mac Ho,5 Colvin M. Redman,1 and Soohee Lee1

Kell (ECE-3), a highly polymorphic blood group glycoprotein, displays more than 30 antigens that produce allo-antibodies and, on red blood cells (RBCs), is complexed through a single disulfide bond with the integral membrane protein, XK. XK is a putative membrane transporter whose absence results in a late onset form of neuromuscular abnormalities known as the McLeod syndrome. Although Kell glycoprotein is known to be an endothelin-3-converting enzyme, the full extent of its physiological function is unknown. To study the functions of Kell glycoprotein, we undertook targeted disruption of the murine Kel gene by homologous recombination. RBCs from Kel(–/–) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin-3-converting enzyme activity and had reduced levels of XK. XK mRNA levels in spleen, brain, and testis were unchanged. In Kel(–/–) mice RBC Gardos channel activity was increased and the normal enhancement by endothelin-3 was blunted. Analysis of the microvessels of tumors produced from LL2 cells indicated that the central portion of tumors from wild-type mice were populated with many mature blood vessels, but that vessels in tumors from Kel(–/–) mice were fewer and smaller. The absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathoC physiological functions of Kell glycoprotein. Am. J. Hematol. 84:492–498, 2009. V 2009 Wiley-Liss, Inc. Introduction In humans, Kell glycoprotein is a highly polymorphic 93 kDa, type II membrane glycoprotein [1]. More than 30 different antigens that have been shown to produce allo-antibodies are associated with Kell glycoprotein [2]. Mutations of the Kell gene resulting in a Kell null phenotype are known, and about 15 null alleles have been reported [3]. Kell null phenotypes are considered normal and no clinical abnormalities associated with null alleles have been documented. On red cells, Kell glycoprotein is linked by a single disulfide bond to its partner protein, XK, a putative membrane transporter that is predicted to traverse the membrane 10 times and expresses the single antigen, Kx, that produces the alloantibody, anti-Kx [4–6]. The expression of Kell glycoprotein and XK are each affected by the absence of the other partner. McLeod red cells, which lack XK, are characteristically acanthocytic, but are also associated with severe depression of all Kell antigens. Red blood cells (RBCs) from Kell null phenotypes also have less XK. A noticeable characteristic of Kell null red cells is that, although these cells have less XK protein, the Kx antigen, as detected serologically, increases. It has been speculated that normally the Kell glycoprotein partially covers the Kx epitope, but that when Kell glycoprotein is absent, Kx is fully exposed [2,7]. Kell glycoprotein is a member of the M13 family of zinc endopeptidases which includes Kell, neutral endopeptidase 24.11 (NEP), two endothelin-converting enzymes (ECE-1 and ECE-2), the product of the PEX gene, and XCE, a protein preferentially expressed in the central nervous system. A common functional feature of the M13 family is that all of the members are involved in the activation, or inactivation, of bioactive peptides. Unlike ECE-1 and ECE-2 which have a preference for big endothelin-1, Kell glycoprotein has a preference for big endothelin-3, which it cleaves to produce
C V 2009 Wiley-Liss, Inc.

bioactive endothelin-3 [8,9]. Substance P and neurokinin A have also recently been shown to be cleaved by Kell, but its affinities for these substrates are relatively low [10]. Endothelins have multiple biological functions, and operate through the activation of two G-protein-coupled receptors, endothelin receptor A (ETA) and endothelin receptor B (ETB), to regulate vasoconstriction, vasodilation, and cell proliferation [11–14]. Endothelin-1 and endothelin-2 binds both ETA and ETB while endothelin-3 preferentially binds to ETB mediating the release of nitric oxide (NO) and prostaAdditional Supporting Information may be found in the online version of this article. Department of Pathology, New York Blood Center, New York, New York; Harvard Medical School, Children’s Hospital Boston, Boston, Massachusetts; 3California National Primate Research Center, University of California– Davis, Davis, California; 4Environmental Health Centre, Ottawa, Ontario, Canada; 5Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts
2 1

Contract grant sponsor: National Institute of Health, a Specialized Center of Research (SCOR) grant in Transfusion Biology and Medicine, HL54459 and an NIH grant, 5R01 HL075716. Conflict of interest: Nothing to report. Xiang Zhu is currently at Peking University Health Science Center, Beijing, People’s Republic of China. Quan Sha is currently at Immunomedics, Inc., Morris Plains,New Jersey. Mac Ho is currently at National Cancer Center, Singapore. *Correspondence to: Soohee Lee, The New York Blood Center, 310 East 67th Street, New York, NY 10065. E-mail: solee@nybloodcenter.org Received for publication 13 May 2009; Accepted 13 May 2009 Am. J. Hematol. 84:492–498, 2009. Published online 18 May 2009 in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/ajh.21453

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Figure 1. Absence of Kell transcripts in spleen and testis, and absence of Kell glycoprotein and endothelin-3-converting activity in RBCs of Kel(–/–) mice compared with WT mice. A: Northern blot of spleen and testis. B: Western blot of RBCs. C: Endothelin-3-converting activity of RBCs. bET-1, bET-2, and bET-3 denote big ET-1, big ET-2, and big ET-3, respectively. Data are expressed as means ± SD of three sets of separate experiments.

Figure 2. Reduction of XK protein in red cells and unchanged XK mRNA level in testis, brain, and spleen of Kel(–/–) mice. A: Reduced level of XK protein, Kel(–/–) lanes of right panel (first 4 lanes, reduced Western blot) and absence of Kell/XK complex in the Kel(–/–) lanes (last 4 lanes, nonreduced Western blot). Left and middle panels are blots stained by Ponceau S to show even loading of the sample proteins before probing the blot by anti-XK (Left 4-lane-panel is reduced and the middle 4-lane-panel is nonreduced samples). The samples are red blood cell ghosts from two WT and two Kel(–/–) mice. B: XK mRNA expression in testis, brain, and spleen of Kel(–/–) and WT mice by Northern blot analysis. Upper panel is probed with 32Plabeled XK cDNA and the lower panel is probed with 32P-labeled b-actin cDNA.

cyclin involving cGMP pathway [15–17]. Endothelin-3 also functions in neovascularization [18], migration of endothelial cells [19,20], guidance of axonal growth [21], promotion of cancers [22–24], and developmental processes of enteric neurons and epidermal melanocytes, affecting migration and differentiation of neural crest-derived cells [25]. Targeted disruption of mouse ETB revealed that homozygous mutants, deficient in ETB, had a phenotype identical to human Hirschsprung’s disease which is associated with defects in the endothelin-3 gene or in ETB. These mice displayed aganglionic megacolon and changes in coat color with abnormal epidermal melanocytes. Mutant mice deficient in endothelin-3 had a similar phenotype, suggesting that endothelin-3 is the natural ligand for the ETB receptor during development [26,27]. Two naturally occurring, recessive mutant mice, piebald-lethal and lethal spotting which have gene defects in ETB and endothelin-3 respectively, were found to display a phenotype identical to that of ETB and ET-3 knockout mice [28,29]. ECE-1 deficient mice also possessed a phenotype similar to ETB and ET-3 deficient mice, indicating that big ET-3 is an important substrate for ECE-1 even though ECE-1 has a preference for big ET-1 and big ET-2. Thus, ECE-1 has overlapping function with Kell glycoprotein [26]. Kell null individuals are not known to exhibit disease symptoms associated with defects in endothelin-3 or in ETB, although Kell glycoprotein has a preference for big endothelin-3. Thus, Kell glycoprotein may not replace the function of ECE-1 in generating Hirschsprung’s disease or other related phenotypes. The expression of Kell glycoprotein is largely limited to erythroid tissues where it is

linked to XK protein and where ECE-1, a homodimer, is not present [8]. Although no clinical abnormalities associated with Kell null alleles have been documented, the full extent of its physiological function is unknown [3]. The expression of Kell glycoprotein in nonerythroid tissues in human and in mouse might be different and only minor amounts in specialized cell types may express Kell glycoprotein. [1,30–32]. Mouse Kell (mKell) [33] is a 110 kDa type II membrane glycoprotein having 80% nucleotide and 74% amino acid sequence identity with human Kell. The mouse Kell gene (Kel) is located on chromosome 6, 20.5 cM and, like its human counterpart, is a single copy gene organized into 19 exons. As in human red cells [6], mKell is disulfide-linked to XK and has endothelin-3-converting enzyme activity. Compared to human Kell glycoprotein, mKell has 8 N-linked sugars rather than 5, an additional extracellular cysteine near the transmembrane region, and a shorter (by 19 amino acids) intracellular N-terminal domain. To study the physiological function of Kell glycoprotein, we deleted, and substituted with a neomycin cassette, a segment of Kel that encodes the zinc binding enzyme active motif, HELLH. Here, we report the generation of the targeted disruption of the mouse Kell gene (Kel) and the biochemical and functional phenotype evaluations of Kell glycoprotein deficient mice. Results Characterization of the Kel(–/–) gene Targeted disruption of Kel was performed by standard homologous recombination procedures as described in

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TABLE I. Blood Pressure and Pulse of Kel(–/–) and WT Mice at 12 Months of age Genotype Kel(–/–), N 5 6 WT, N 5 6 Systolic ± SE 100.3 ± 5.84 109.1 ± 6.11 P 5 0.320a Pulse ± SE 658.7 ± 61.0 751.3 ± 16.8 P 5 0.005a Systolic success ± SE 15.33 ± 1.28 18.17 ± 0.48 P 5 0.065a

a

t-test P-value at 0.05.

TABLE II. Red Cell Indices of Kel(–/–) and WT mice Attribute HGB (g/dL) MCV (fL) MCH (qg) MCHC (g/dL) CH (qg) HDW (g/dL) ReticP RBC (x103 cells/lL) HCT (%)
a

WT ± SD N 5 25a Kel(–/–) ± SD N 5 17a P-valuea at 0.05 10.37 46.98 13.51 28.74 13.40 2.05 2.93 7.64 35.78 ± ± ± ± ± ± ± ± ± 0.83 0.69 0.39 0.66 0.20 0.11 0.56 0.57 2.70 10.85 48.43 14.23 29.33 13.81 2.16 3.35 7.65 36.95 ± ± ± ± ± ± ± ± ± 0.50 0.88 0.50 0.81 0.28 0.15 0.60 0.43 1.90 0.11 <0.0001 <0.0001 0.024 <0.0001 0.013 0.064 0.96 0.23

Figure 3. RT-PCR of spinal cord of WT and Kel(–/–) mice.Lane 1, 1 kb DNA ladder; lanes 2–5, Kel(–/–); lanes 6–9, WT. Lanes 2 and 6 are for Kel RT-PCR (351bp) showing the presence of 351-bp band only in WT (arrow). Lanes 3 and 7 are XK (373 bp). Lanes 4 and 8 are the positive control, G3PDH (456 bp).

Materials and Methods and summarized in Supporting Information Fig. 1. Examples of results of the screening are shown in Supporting Information Fig. 2A. Heterozygous DNA showed the presence of both bands for WT and Kel knockout alleles when cut with BamHI or EcoRV (last lanes of A, upper and lower panel). Of 5 chimeric mice, 2 exhibited germ line transmission of the Kel targeted allele. Results of PCR screening of genomic tail DNAs are illustrated in Supporting Information Fig. 2B, which shows 349 bp for wild type (lane 2), 941 bp for Kel(–/–) (lane 5), and both bands for Kel(1/–) (lanes 6 and 7). Lack of Kell mRNA and Kell glycoprotein in Kel(–/–) mice Northern blot analysis of Kell mRNA in spleen and testis demonstrated the absence of normal Kell mRNA (2.5 kb) in Kel(–/–) mice (Fig. 1A). The low molecular weight bands seen in Kel(–/–) mice indicate the presence of truncated Kell mRNA (which would be detected by the probe used). This was confirmed by the presence of the predicted RTPCR product derived from the nontargeted area of the Kel gene (data not shown). Western blot analysis of RBC membrane proteins from Kel(–/–) mice showed the absence of Kell glycoprotein (Fig. 1B, right panel, second lane). As shown in Fig. 1C, there was also a lack of endothelin-3converting enzyme activity by Kel(–/–) red cells. Heterozygous Kel(1/–) red cells showed decreased activity compared to wild-type red cells (Fig. 1C). Together, these results demonstrated that the Kel knockout successfully disrupted Kell glycoprotein expression and removed its enzymatic activity. Red cell XK protein is reduced but XK mRNA is unchanged in Kel knockout mice XK protein was decreased in Kel(–/–) red cells compared to wild-type red cells (Fig. 2A, right panel, reduced western blot, Kel(–/–) lane). Kell/XK complex is also absent from Kel(–/–) RBCs (Fig. 2A, right panel, nonreduced, Kel(–/–) lane). Panels stained with Ponceau S show even loading of samples. XK protein is reduced in Kel knockout red cells as shown in 2A, but mRNA levels are unchanged in testis and spleen where Kell glycoprotein and XK are coexpressed (Fig. 2B). The XK mRNA level in brain, where little or no Kell glycoprotein is expressed, is also unchanged. Expression of Kell in mouse spinal cord Detection of expression of Kell in mouse brain has been inconclusive and it has been suggested that there may be

Statistical significance of genotype effect was analyzed by two-way ANOVA on the two sets of data: one set contained 18 WT and 9 Kel(–/–) mice, and the second set contained 7 WT and 8 Kel(–/–) mice. HGB, hemoglobin concentration; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CH, red cell hemoglobin content; HDW, hemoglobin distribution width; ReticP, reticulocyte percentage; RBC, red blood cell count; HCT (%), hematocrit percentage.

species differences between mouse and human [31]. As determined by in situ hybridization, the expression of mouse Kell was high in erythroid, but not in nonerythroid tissues. By RT-PCR, mouse Kell mRNA was detected in testis but not in muscle or brain. However, using Kel(–/–) tissue as a negative control, RT-PCR of spinal cord from wild-type mouse indicated weak expression of Kell transcripts in this tissue (Fig. 3). Lanes 5 and 9 (Fig. 3) show that a weak RT-PCR band (235 BP) of an erythroid specific gene product, glycophorin A (GPA), was present as well, which may indicate contamination by erythroid cells, but expression of GPA in nonerythroid cells is also possible as shown in the GPA gene (GYPA) expression profiles available at GeneCards site [34–36]. Blood pressure, pulse, and ECG analysis The systolic blood pressure and pulse of 6 WT and 6 Kel(–/–) mice were measured. As shown in Table I, systolic blood pressure was not significantly different between WT and Kel(–/–) mice, but pulse of Kel(–/–) mice was significantly slower than among WT mice (P < 0.005). ECG analysis showed that the P-Q and P-R intervals were significantly decreased in Kel(–/–) mice than in WT mice (P < 0.05) suggesting that there may be some effect coming from altered sympathovagal balance probably involving changes in NO signaling [37,38]. Hematological analysis of Kel(–/–) mouse blood Two sets of analyses done on two different dates were combined and analyzed by two-way ANOVA. There were small but significant increases in MCV, MCH, CH, MCHC, and HDW in Kel(–/–) RBCs compared to WT RBCs (Table II). However, the biological significance of these small increases is not known. Measurement of channel activity of RBCs and the effect of ET-3 Because Kell glycoprotein activates ET-3 and ET-3 is known to modulate Ca11activated K1 channels (Gardos channel) [39] it was important to investigate Gardos channel activity in Kel(–/–)RBCs. Therefore, we measured

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TABLE III. Counting of CD31 Positive Microvessels in the Central Part of Tumors Age MCV counts/ tumor tumor tumor tumor tumor WT ± SE (Na) 31.42 30.20 31.33 23.75 ± ± ± ± 4.68 6.52 2.73 4.23 (12) (5) (3) (4) Kel (–/–) ± SE (Na) t-test P-value 10.00 17.88 17.80 5.333 ± ± ± ± 1.60 5.51 3.40 4.33 (9) (8) (5) (3) 0.0012b 0.18 0.034b 0.031b

5 months 2-week 3-week 12 months 2-week 3-week
a b

N 5 number of tumors counted. P value  0.05.

Figure 4. Comparison of Gardos channel activity of RBCs of Kel(–/–) and WT mice and the effect of ET-3. Gardos channel activity is defined by ChTx-sensitive K1 influx measured by 86Rb uptake. Data are expressed as means ± SD of 5 to 7 separate experiments in duplicate determinations. Statistical analysis was evaluated by student’s t-test: *P < 0.05, n 5 5 to 7.

the maximal activity of Gardos Channel in freshly isolated erythrocytes from Kel(–/–) mice compared to WT. We found that the deletion of Kell glycoprotein induced an increase in Gardos channel activity under maximal conditions (Fig. 4). These results suggested that Kell glycoprotein is involved in the regulation of the Gardos channel. To determine if the regulatory effect of ET-3 on the Gardos channel, as observed previously [39], was disrupted by Kell deletion, we measured Gardos channel activity in the presence of ET-3. As shown in Figure 4, the activation effect of ET-3 on the Gardos channel was blunted by the deletion of Kell, suggesting that Kell glycoprotein plays an important role in the interaction of ET-3 with the Gardos channel, possibly by facilitating receptor-ligand interactions or activation of the signal transduction pathway involving ET-3 and ETB. It is known that the functional ETB is present on RBCs [39,40]. Analysis of microvessel formation in Lewis lung carcinoma tumors In two sets of experiments, tumors grown in 5- and 12-month-old mice for 2 weeks and 3 weeks were collected, weighed, and measured. There was no difference in size and weight of tumors from WT and Kel(–/–) mice. The number of microvessels at the boundary (periphery) of tumors in WT and Kel(–/–) mice did not differ. However, the number of microvessels, identified by CD31 immunostaining, in the central part of 2-week tumors was significantly lower in Kel(–/–) mice than in WT in both age groups (P 5 0.0012 for 5-month-old mice and P 5 0.0340 for 12-monthold mice). In Kel(–/–) mice 3-week tumors from 12-monthold mice exhibited significantly reduced numbers of microvessels (P 5 0.031). Similar tumors from 5-month-old mice exhibited reduced numbers of microvessels, but the difference was not significant (P 5 0.1835) (Table III). Examples of immunohistologic examination, shown in Fig. 5, illustrate that in WT mice the central part of the tumors are rich in microvessels and the vessels appear to be mature (Fig. 5A,C,E,G). In contrast, in Kel(–/–) mice, the microvessels in the central part of the tumors are fewer and smaller (Fig. 5B,D,F,H). Mild motor dysfunction phenotype of Kel(–/–) mice No severe neurological symptoms (stereotypy, seizures, or autonomic signs) were recorded in the Functional Observational Battery. Normal responses to handling, visual, tactile, and auditory stimulation were seen. Scores for posture and the simple reflexes were in the normal range. However, in observational scoring of gait abnormality, 1 of 6 WT mice received an abnormal score as compared to 3 of 6 Kel (–/–). This difference was not statistically significant.

Figure 5. Microvessels labeled by CD31 in the central part of 2-week tumors in WT (A, C, E, and G ) and Kel(–/–) mice (B, D, F, and H ). Magnification: A, B x40; C, D x100; E, F x200; G, H x400.

Similarly, grip strength, stride length, and wire hang tests were not consistently affected by lack of Kell. However, a decline in forelimb strength was observed in Kel(–/–) mice from 12 to 16 months of age P 5 0.014), but not in WT mice (P 5 0.55). Further, mice lacking Kell had greater hindlimb foot splay in the drop test over all three time points (6, 12, and 16 months of age) by RMANOVA (P 5 0.012) (Table IV). On the rotarod test, mice lacking Kell generally had more falls during training and a shorter time to fall during testing at increasing speeds, but the group effect approached significance only for the second trial at the highest speed (44 rpm) when the mean time to fall was 13 ± 3 sec in the WT mice and 6 ± 3 in the Kel(–/–) mice ( P 5 0.054). There were no effects of genotype on measures of spontaneous activity (horizontal and vertical movements or distance traveled).

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TABLE IV. Grip Strength, Wire Hang, Stride Length, and Foot Splay in the Drop Test of Kel(–/–) Mice Compared With Wild- Type Mice Age Age

6 months 12 months 16 months 6 months 12 months 16 months Grip WT Kel(–/–) Forelimb grip strength ± SD (g) 0.85 ± 0.13 0.93 ± 0.14 0.80 ± 0.11 0.78 ± 0.16 0.76 ± 0.13 0.72 ± 0.11 Hindlimb grip strength ± SD (g) 0.59 ± 0.10 0.50 ± 0.11 0.49 ± 0.17 0.51 ± 0.19 0.49 ± 0.13 0.46 ± 0.19

Wire hang WT Kel(–/–) Stride WT Kel(–/–) Drop

Time to fall ± SD (sec) 27 ± 15 18 ± 6 29 ± 24 30 ± 11 21 ± 13 24 ± 5 0.67 ± 0.82 1.33 ± 0.81

Number of falls ± SD 1.33 ± 0.51 1.67 ± 0.82 2.00 ± 0.89 1.67 ± 1.03

Forelimb stride length ± SD (mm) 64 ± 8 68 ± 5 69 ± 7 71 ± 6 66 ± 7 69 ± 7

Hindlimb stride length ± SD (mm) 73 ± 6 72 ± 5 68 ± 10 71 ± 4 66 ± 7 71 ± 13

Forelimb drop foot splay ± SD (mm) 16 ± 3 19 ± 3 20 ± 3 19 ± 3 18 ± 2 17 ± 2

Hindlimb drop foot splay ± SD (mm)a 27 ± 4 32 ± 4b 32 ± 3 39 ± 3c 31 ± 2 41 ± 2d

WT Kel(–/–)
a

Significance of the effect of Kel genotype on hindlimb foot splay in the drop test over all three time points (6, 12, 16 months) by RMANOVA, P 5 0.012; 6 month time point. b P 5 0.40; 6 month time point. c P 5 0.036; 12 month time point. d P 5 0.021; 16 month time point.

Discussion Here, we report analysis of mice carrying targeted disruption of the Kel gene. We found that RBCs from Kel(–/–) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin-3-converting enzyme activity, and had reduced levels of XK as is seen in Kell null humans [3]. XK mRNA levels in spleen, brain, and testis, however, were unchanged. In Kel(–/–) RBCs, Gardos channel activity was increased but the modulation of the channel by endothelin-3 was blunted. The central portions of tumors from WT mice were populated with many mature blood vessels in comparison with those of Kel(–/–) mice which were fewer and smaller. In behavioral tests, the absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. Reduction of XK levels in erythroid tissues may be due to increased intracellular proteolytic degradation of XK. In transfected cells expressing both Kell glycoprotein and XK, the Kell/XK complex is formed early in the endoplasmic reticulum during transport to the cell surface, and although Kell glycoprotein is not absolutely required for XK transport to the cell surface, in Kel(–/–) cells the lack of Kell glycoprotein may hamper XK transport, resulting in XK being more readily degraded [41]. Thus, XK may be more susceptible to degradation as a free molecule than it is in its normal form as part of the disulfide bonded Kell/XK complex. The expression of Kell glycoprotein in humans was originally thought to be limited to erythroid tissues, but later reports suggested that Kell glycoprotein is also expressed in testis and in other nonerythroid tissues including brain and skeletal muscle [1,30,32]. In mice, analysis of expression of Kell glycoprotein by in situ hybridization revealed that Kell glycoprotein expression is limited to erythroid tissues with a small amount in testis [31]. The earlier results indicating the presence of Kell glycoprotein in nonerythroid tissues in humans may have been partly due to the presence of residual blood in the tissues examined. Our current RT-PCR studies indicate that Kell transcript is present in

spinal cord, and may be expressed only in some specialized cells in nervous tissues. There was no significant difference in plasma endothelin3 levels between Kel(–/–) and WT mice (data not shown). On red cells, Kell glycoprotein may function as an endothelin-3-converting enzyme only when the substrate, big endothelin-3, is available in sufficient concentration and the local pH is optimal for Kell enzymatic activity. The substrate concentration (big endothelin-3) in plasma is in the picomolar range, which is much less than the Km value, which is in the micromolar range [8]. We hypothesize that Kell glycoprotein, as an endothelin-3-converting enzyme, may have a limited role in normal physiology, but may function when red cells are carried to pathological sites, such as tumors, where there is a suitable microenvironment for its activation. Kell null individuals, like Kel(–/–) mice, have no easily detected abnormalities and parameters such as lower heart rates and decreased motor activities. If present, such abnormalities and parameters may go undetected in a normal clinical examination. When Kell glycoprotein is absent the amount of XK is reduced and the Kell/XK complex no longer exists [4,6]. XK may play a role in regulating K1 flux and the reduction in XK in Kel(–/–) mice may account for the difference in Gardos channel activity seen in Kel(–/–) mice. It is also possible that XK function differs depending on whether it exists in a complex with Kell glycoprotein or as a monomer. In this respect, Kell glycoprotein may be a regulator of XK ion transport function. The increased number of microvessels in the central part of tumors in WT mice compared to Kel(–/–) mice indicates that Kell glycoprotein may be involved in the formation of blood vessels or in the interaction of vascular endothelial cells with tumor cells. The observation that the center and not the periphery of the tumor raised in Kel(–/–) mice had less microvessel formation suggests that in that microenvironment, Kell, by it’s endothelin-3-converting enzyme activity, is involved in neovascularization in a manner that is nonoverlapping with the function of ECE-1 [26]. We expect that the center of the tumor may be hypoxic and acidic due to anaerobic glycolysis. The optimum pH for Kell enzyme activity is acidic, pH 6 [8], and in addition the Kell promoter has HIF1 (Hypoxia-Inducible Factor 1) binding sites (nt -829 and 1655) although we do not know if they are functionally active. If they are, the expression of the Kell gene is hypoxia inducible. These conditions are beneficial for Kell enzymatic function and for activating endothelin-3 locally. As noted earlier, Kell glycoprotein may also be involved in the interaction of endothelial cells with tumor cells but we did not detect Kell expression in endothelial cell lines (HUVEC) or HUVSM by RT-PCR (data not shown), although, according to the Transcription profiling of human cell lines and tissues of the EBI data base, Kell transcripts were detected in CD105 (Endoglin) positive endothelial cells (samples 32 and 106, Array Express Warehouse 8.10). CD105 is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells [42]. We did not detect severe motor function pathology in mice lacking Kell. However, some motor characteristics could be identified as part of the phenotype of mice lacking the Kell gene. Mice lacking Kell glycoprotein demonstrated wider hind limb landing foot splay in the drop test, and, although statistical significance was not reached, in rotarod testing generally had more falls during training and a shorter time to fall during testing at increasing speeds. In conclusion, the phenotypes in Kell knockout mice suggested possible physiological functions of Kell glycoprotein in heart, red cell ion transport, neovascularization in tumors, and motor function. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not

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be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathophysiological functions of Kell. Materials and Methods
Targeting vector. Targeted disruption of Kel was accomplished by homologous recombination using a targeting vector constructed in pBluescript using a Kel clone that has a 12.9 kb EcoRI-EcoRI fragment which contains exons 11 to 19. The Kel clone was selected by screening the Mouse 129 SvJ genomic library (Stratagene, La Jolla, CA). A 3.2 kb segment of Kel, spanning a region from exon 11 to part of exon 16 (HindIII to EcoRV fragment) and which includes the region encoding the zinc binding, enzymatically active site of Kell glycoprotein, was replaced with a 1.6 kb cassette containing the neomycin-resistance gene (Supporting Information Fig. 1). A 2.7 kb segment of intron 10 was placed in the 5’ short arm of the construct and a 7 kb fragment downstream of intron 16 was placed in the 3’ long arm. The EcoRV site at the 5’ end of the long arm was destroyed and a BamHI site at the 3’ end of short arm was created when the neomycin gene was inserted, resulting in the generation of a 15 kb band when cut by EcoRV and a 3.7 kb band when cut by BamHI, compared to 8 and 9 kb bands, respectively, in the wild type allele. Generation of mice carrying the targeted allele. RW-4 ES cells derived from the 129/SvJ mouse strain were electroporated with the linearized targeting vector and the positive cells were selected using G418 in the culture medium. Homologous recombinants were identified by Southern blot analysis of EcoRV or BamHI cut genomic DNA. Chimeras were produced using morula aggregation technology [43] (GenomeSystems, Inc). The germ line transmitted heterozygous Kel knockout [Kel(–/–)] mice were selected by mating chimeras (male and female) with C57BL/6 mice. The genetic background of the knockout mice was made fully congenic by back-crossing the hybrids with C57BL/6 for at least 10 generations. Finally, homozygous Kel(–/–) mice were generated by mating the Kel(1/–) mice. Screening of Knockout mice by Southern blot and genomic DNA PCR, and characterization by Western blot. Southern blot. DNA obtained from mouse tails was cut with BamHI or EcoRV followed by agarose gel electrophoresis. The separated DNA was blotted on Zeta-Probe GT Blotting Membrane (Bio-Rad). BamHI cut DNA was hybridized with a 600bp 5’ end probe that distinguishes the wild type Kel gene (9 kb band) from the knockout allele, Kel(–/), (3.7 kb band). EcoRV cut DNA was hybridized with a 700 bp 3’ end probe that gives an 8 kb band for the wild type allele and a 15 kb band for Kel(–/) allele. The size and the location of the probes are described in the construct map (Supporting information Fig. 1). PCR. The following primers were used for screening the tail genomic DNA by PCR. Kel WT:(Min10FA) 5’-GCCTGAGTTGTTTTCCTATCCCTTGG-3’ (Min11RA) 5’-GCACACCACAAAGAAGTATACACTTC-3’ The resulting amplicon is 349 bp in size Kel KO: (3’probF) 5’-AGGAGTCAGTGGCAGTACCAGATTCTAG-3’ (NeoS) 5’-GGATGCGGTGGGCTCTATGGCTTCTG-3’ The resulting amplicon is 2.8 kb in size. This primer set was used to confirm the integrity of the targeted gene at the 5’ end recombination site. Subsequently, the following primer set, which amplifies a shorter segment with an improved signal was used for identification of the Kel(–/) allele. mKel-WTKO-1F: 5’-GTGCCTGAGTTGTTTTCCTATCCCTTGG-3’ mKel-KO-1R: 5’-CATTCGACCACCAAGCGAAACATCGCAT-3’ The resulting amplicon is 941 bp in size. Western blot. Blood was collected into EDTA-containing tubes and red cell ghosts were prepared with low salt buffer (5 mM sodium phosphate buffer, pH 7.4 containing 10 U/mL trasylol). The ghosts were dissolved in 23 sample buffer with or without 1 mM DTT and the sample was separated in 10% SDS-PAGE. Kell glycoprotein was probed with a rabbit antibody to a peptide of mouse Kell (mKell 358-393), and XK was probed with rabbit antibody to a mouse XK peptide (mXK 404443) using a western blot kit (SupersignalTM, Pierce). Determination of endothelin converting activity of Kell glycoprotein. Endothelin converting activities of RBCs of Kel(–/–), Kel(1/–), and WT mice were analyzed using the ET-1 enzyme immunoassay (EIA) Kit (Cayman Chemical, Ann Arbor, MI) following the company protocol as described previously [8]. RT-PCR. The RT-PCR was carried out as described previously [31]. Physiologic examination of heart function. Electrocardiogram and blood pressure. Six conscious 1-year-old female mice underwent a single continuous ECG test at the Jackson Laboratory (West Sacramento, CA) using the noninvasive AnonyMOUSE ECG screening system (Mouse Specifics, Inc). Systolic blood pressure testing was also conducted over 30 consecutive cycles of cuff inflation and deflation (10 preliminary and 20 test measurements) using the Visitech BP-2000 blood pressure analysis system. Hematological analysis. Blood samples (25 lL) from 25 male WT and 17 male Kel(–/–) mice of 153 to 173 days of age were collected by retro-orbital sinus bleeding using heparinized capillary tubes. Blood was mixed with 100 lL RPMI and further diluted with 100 lL of 1% BSA in RPMI before analysis. The tests were performed in two groups on two different days. Group one contained 18 WT and 9 Kel(–/–) mice and group two contained 7 WT and 8 Kel(–/–) mice. Complete blood analysis was performed using the Advia 120 Hematology System (Bayer, Tarrytown, NY). Measurement of Gardos channel activity of RBCs and the effect of ET-3. Erythrocyte preparation. The procedure for the preparation of erythrocytes was followed as described previously [39]. Measurement of 86Rb influx. The measurement of 86Rb influx was carried out by a previously described method [39]. Briefly, the influx medium contained 1 mM ouabain (Sigma chemical, St. Luis, MO) 10 mM Tris-MOPS, pH 7.4 (228C), 10 lM bumetanide, and 10 lCi/ml 86Rb with and without 700 nM ET-3 (Sigma chemicals) and 50 nM charybdotoxin (ChTX, Sigma chemical). Preincubations with ET-3 were carried out for 15 min at 378C in an isotonic Na-media. Free ionic Ca21 in the influx media was buffered to 7 lM with 1 mM citrate buffer. Ca21 concentration was calculated using the dissociation constants for citrate and correcting for ionic strength in the presence of 0.15 mM MgCl2 at pH 7.4. At time 0 min, A23187 ionophore (5 lM) (Calbiochem-Novabiochem Corp., La Jolla, CA) was added and aliquots were removed at 2 and 5 min and the erythrocyte-associated radioactivity was counted as described previously [40]. Tumor growth and neovascularization. The highly tumorigenic and weakly metastatic Lewis lung carcinoma cells (LL/2 cell line originally derived from a C57BL/6 mouse, ATCC) were injected subcutaneously on the backs of Kel(–/–) and WT mice (106 cells/ mouse). Before injection, the possible presence of adventitious and endogenous viruses in the cultured LL/2 cells was assessed by MAP (Mouse Antibody Production) test at the Diagnostic laboratory of Charles River Laboratories. By 2 or 3 weeks following injection, the tumors reached 1 cm3, at which point the mice were euthanized and the shape, weight, and vascularization of the tumors were measured. The formation of vascular structures in and around the tumor was examined by light microscopy. Two sets of experiment were performed, the first using 12month-old mice and the second using 5-month-old mice. Tumor tissues were fixed in IHC Zinc Fixative (Formalin-Free) solution (BD Biosciences, San Jose, CA) for not more than 24 hr, embedded in paraffin blocks and sectioned (5–6 lm thickness). Immunohistochemical staining of tissue sections for CD31 (PECAM-1). Tissue sections were examined for neovascularization by immunostaining with anti-CD31 (BD biosciences) using the TSATM Biotin System (Perkin Elmer, Waltham, MA) in conjunction with NovaREDTM (Vecter Labs, Orton Southgate, Peterborough, UK) visualization reagent following the manufacturer’s protocol. Antigenic epitopes were retrieved by digestion with Trypsin (Sigma, St. Louis, MO) at 378C, for 10 min. Endogenous peroxidase was blocked with 3% H2O2 for 5 min. The area selection for counting the microvessels was performed, first, by viewing the tumor with a low power objective lens (43) to find the center part of the tumors and, subsequently, by randomly selecting 1–3 fields depending on the tumor size with a higher power objective lens (203) . Counting of microvessels was performed at 2003 magnification using SPOT 4.1 software.The per-spot values from each tumor were averaged. The averaged value was used in the statistical analysis to represent one tumor. Evaluation of motor function. Design and testing schedule. Six WT and 6 Kel(–/–) male mice were assessed at 6 and 12 months of age with grip strength, ataxia, wire suspension, and spontaneous motor activity tests. Additionally, an observational assessment, the Functional Observational Battery, was conducted monthly. At 18 months of age, mice were added to the groups [n 5 17 Kel (1/1), n 5 15 Kel(–/–)] for a more challenging motor test, the incremental speed rotarod. Tests were performed at the Murine Behavioral Assessment Laboratory of the University of California-Davis using standardized protocols and have been described previously [44–47]. Briefly, the Functional Observational Battery is a 22-item neurological screen for abnormal reflexes, motor patterns, seizure activity, and autonomic signs. For the ataxia test, stride length is measured as the distance between successive

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paw placements as the mouse walks down a narrow path, and landing foot splay in the drop test is recorded as the distance between paws when the mouse is dropped from a height of 16 cm. Grip strength is measured as the force required to break the grip reflex. For wire suspension, the mouse grasps a 2 mm wire and has 60 sec to make its way to a side support. The spontaneous activity tests used beam break technology to track movement in smaller (10 x 10 inch) and larger (16 x 16 inch) arenas for shorter (90 min) or longer (20 hr) evaluation periods. Rotarod testing included 3 days of training at 24 rpm followed by two 60-sec trials at speeds of 8, 16, 24, 30, 33, and 44 rpm [48]. Statistical analysis. All statistical analysis except data for motor function evaluation was performed by Student’s t-test (unpaired two-tail analysis) or two-way ANOVA using GraphPad Prism software (La Jolla, CA). For the motor function evaluation data, a single factor ANOVA was conducted with Kel genotype as the independent variable. Repeated measures ANOVA (RMANOVA) was used for the sequential tests. If homogeneity of variance across groups was not observed, the Welch ANOVA, which does not assume equal variance, was used. All statistical analyses were performed with JMP software (SAS, Cary, NC).
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Acknowledgments The authors thank the staff of the Nucleic Acid Analysis Laboratory of the New York Blood Center for DNA sequencing. They also thank Dr. Eric C. B. Milner of BioMedical Science Writers, LLC, for helping in editing the manuscript. References
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