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Intracellular Assembly of Kell and XK Blood Group Proteins

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Intracellular Assembly of Kell and XK Blood Group Proteins View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1461 (1999) 10^18 www.elsevier.com/locate/bba Intracellular assembly of Kell and XK blood group proteins David Russo, Soohee Lee, Colvin Redman * Lindsley F. Kimball Research Institute, The New York Blood Center, 310 East 67 Street, New York, NY 10021, USA Received 22 June 1999; accepted 13 August 1999 Abstract Kell, a 93 kDa type II membrane glycoprotein, and XK, a 444 amino acid multi-pass membrane protein, are blood group proteins that exist as a disulfide-bonded complex on human red cells. The mechanism of Kell/XK assembly was studied in transfected COS cells co-expressing Kell and XK proteins. Time course studies combined with endonuclease-H treatment and cell fractionation showed that Kell and XK are assembled in the endoplasmic reticulum. At later times the Kell component of the complex was not cleaved by endonuclease-H, indicating N-linked oligosaccharide processing and transport of the complex to a Golgi and/or a post-Golgi cell fraction. Surface-labeling of transfected COS cells, expressing both Kell and XK, demonstrated that the Kell/XK complex travels to the plasma membrane. XK expressed in the absence of Kell was also transported to the cell surface indicating that linkage of Kell and XK is not obligatory for cell surface expression. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Kell; XK; Blood group; Red cell; Protein assembly 1. Introduction and the Kell blood group system is important in transfusion medicine since Kell antibodies can elicit Kell is a 93 kDa type II membrane glycoprotein [1] severe reactions if incompatible blood is transfused and XK a 444 amino acid integral membrane protein [5,6]. Kell antibodies can also suppress erythropoiesis that is predicted to span the membrane 10 times [2]. and cause anemic disease in newborn infants [7^9]. Both Kell and XK proteins display blood group anti- The di¡erent Kell antigens are due to point muta- gens and on human red cell membranes Kell and XK tions in KEL leading to single amino acid changes in are linked by a disul¢de bond [3,4]. Kell is highly the extracellular domain of the glycoprotein [10^15]. polymorphic, and over 20 di¡erent antigens are rec- XK on the other hand, carries a single blood group ognized as part of the Kell blood group system. antigen, termed Kx, which is present in all red cells Some of the Kell antigens are strong immunogens except in the rare McLeod phenotype. McLeod red cells have aberrant shape, appearing as acanthocytes, and together with lack of XK there is a marked Abbreviations: NEP, neutral endopeptidase; ECE, endothelin diminution of Kell protein and all Kell antigens are converting enzyme; ER, endoplasmic reticulum; SDS, sodium markedly weakened. Lack of XK is associated with dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; disease and most McLeods exhibit a late onset form PMSF, phenylmethyl sulfonyl £uoride; TPCK, N-tosyl-L-phenyl- alanine chloromethyl ketone of muscular dystrophy, cardiomyopathy, and nerve * Corresponding author. Fax: +1-212-879-0243; degeneration [5,6,16,17]. E-mail: [email protected] Expression of wild-type and mutant recombinant 0005-2736 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S0005-2736(99)00148-0 BBAMEM 77694 25-10-99 D. Russo et al. / Biochimica et Biophysica Acta 1461 (1999) 10^18 11 Kell and XK proteins has indicated that Kell Cys72, peptide representing the N-terminal domain of Kell which is close to the membrane spanning domain, is protein (amino acids 2 to 31) was synthesized by the linked to XK Cys347 located in a small `loop' which Microchemistry Laboratory of the New York Blood contributes the ¢fth extracellular XK domain [18]. Center. Polyclonal antibodies were produced in rab- Although Kell and XK exist on red cell membranes bits by injection of the synthetic peptides mixed with as a disul¢de-bonded complex, association of the two Freunds adjuvant and the antisera were a¤nity pu- proteins is not needed for presentation of Kell blood ri¢ed. group antigens. Expression of Kell protein in a vari- ety of transfected cells indicates that Kell can be 2.2. Construction of expression vectors transported to the plasma membrane without co-ex- pression of XK and the recombinant protein ex- XK cDNA was a generous gift from Drs. M. Ho presses Kell surface antigens [15,19]. Likewise, Kx and A.P. Monaco, Imperial Cancer Research Fund antigen is present in Ko (null) red cell membranes Laboratories, Institute of Molecular Medicine, John which have little, or no, Kell protein [5,6]. It is not Radcli¡e Hospital, Oxford, England. Two pRC/ known, however, whether the association of Kell and CMV expression vectors were constructed. One vec- XK is needed for red cell functions, since the com- tor contained only XK cDNA and the other had plete physiological roles of the two proteins remain both XK and Kell cDNAs. For the vector containing to be determined. Kell protein has close structural only XK, the cDNA was placed in pRc/CMV (Invi- and sequence homology to the M13 or neprilysin trogen, Inc.) in HindIII and XbaI cloning sites. For sub-family of zinc endopeptidases [15] which include the vector containing both XK and Kell, the Kell neutral endopeptidase 24.1 (NEP), endothelin con- cDNA was modi¢ed to contain a Kozak sequence verting enzyme (ECE) and the product of the PEX (GCCGCCACC) [26] before the ATG initiation co- gene [20^24]. The members of the M13 family of don. The modi¢ed Kell cDNA and XK cDNA were membrane zinc endopeptidases are involved in the placed in tandem, each with its own CMV promoter activation and inactivation of biologically active pep- and adenylation signal, into pRc/CMV as previously tide hormones. Recently we determined that the ex- described. tracellular domain of Kell protein, like ECE-1, acti- vates the endothelins, but that unlike ECE-1 Kell 2.3. Transient transfection of COS cells preferentially activates endothelin-3 rather than en- dothelin-1 [25]. Although absence of XK is associ- COS-1 cells (ATCC CRL 1650) were transfected ated with red cell acanthocytosis and with muscle by electroporation using a Gibco Cell Porator with and nerve disorders and its primary structure has an electrical pulse of 250 V and 330 WF. About features that suggest that it may be a membrane 2U107 cells were suspended for 15 min in 1 ml of transporter [2], its function remains to be deter- Dulbecco's phosphate-bu¡ered saline with 10 Wgof mined. plasmid DNA prior to electroporation. The cells To study the mechanism of Kell/XK assembly we were allowed to recover in ice for 10 min and were have, using co-expression of Kell and XK in COS seeded at about 5U106 cells on 100 mm plastic Petri cells, determined the biosynthesis, site of assembly dishes (Falcon, Becton Dickinson, Franklin Lakes, and intracellular transport of Kell and XK proteins. NJ, USA). The cells were cultured in 10 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum and grown to con£uence, usually in 2. Materials and methods 2 days. 2.1. Antibodies 2.4. Metabolic labeling of transfected COS cells with 35 L-[ S]methionine A 42-mer synthetic peptide, corresponding to the predicted second extracellular loop of XK was pre- Con£uent COS-1 cells were washed with phos- pared by Synpep, Deblin, CA, USA and a 30-mer phate-bu¡ered saline and incubated at 37³C for 30 BBAMEM 77694 25-10-99 12 D. Russo et al. / Biochimica et Biophysica Acta 1461 (1999) 10^18 min in 1 ml of L-methionine-free Dulbecco's minimal from the cytoplasmic domain of Kell, were added essential medium (Life Technologies, Inc.) containing and incubated overnight at 4³C. The immune com- 35 0.5 mCi of L-[ S]methionine (NEN Research Prod- plex was isolated with protein A-Sepharose, the pro- ucts, Boston, MA, USA, speci¢c activity 1110 Ci/ teins eluted with SDS bu¡er (0.125 M Tris-HCl, pH mmol). The cells were then washed twice with phos- 6.8, 1% SDS, 5% glycerol) containing 4 M urea and phate-bu¡ered saline and incubated with growth me- separated on 9% polyacrylamide gels [18,30] or pre- dium for various periods of time. casted 4^12% Tris-glycine gradient gels (Novex, San- diego, CA, USA). 2.5. Cell fractionation The immune complex was treated with endonu- clease-H (New England BioLabs Inc., Beverley, About 108 transfected COS cells were lysed with MA, USA) to remove the high mannose chitobiose 2 ml of ice-cold water for 5 min and the cells further core from glycoproteins [31] as recommended by the disrupted with a loose-¢tting Dounce homogenizer. manufacturer. The control samples that were not The cell suspension was adjusted to 0.25 M sucrose treated with endonuclease-H underwent the same and centrifuged at 500Ug for 10 min to remove un- procedure except that the enzyme was not added. broken cells, debris and a nuclear fraction. The supernatant fraction was adjusted to 1.38 M sucrose 2.8. Two-dimensional gel electrophoresis and overlaid over 2 M sucrose. A discontinuous su- crose gradient of 1.1 M, 0.6 M and 0.25 M sucrose In the ¢rst dimension, radioactive proteins present was then applied and centrifuged in a Beckman SW in the immunoprecipitate obtained with antibody to 40.1 rotor at 105 000Ug for at least 3 h.
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