Molecular basis of the K:6,‐7 [Js(a+b−)] phenotype in the Kell blood group system

Lee, S.; Wu, Xu; Reid, Marion E.; Redman, Colvin M.

doi:10.1046/j.1537-2995.1995.351096026362.x

Cited by 46 publications

(29 citation statements)

References 14 publications

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“…2). This result is in accordance with previously published studies that have shown that K1 antigenic sites were found in equivalent quantities to K2 sites on heterozygous red cells (50), suggesting that, despite its modified sequence and as demonstrated here, polysaccharide content, K1 still attains a conformation validated within the ER compartment, a conformation that, at the cell surface, possesses superior antigenicity than that of Kell K2 (29). Thus it appears that there may be many "correct" conformations to one protein or that subtle conformational changes cannot be distinguished within the ER, even though both antigens may display different retention times within this compartment (Fig.…”

Section: Discussionsupporting

confidence: 94%

“…Among the identified point mutations of the Kell system so far, one may affect such a process, that of Kell K1, which is defined by a C to T base substitution in exon 6 leading to the replacement of a threonine by a methionine residue (T193M) within a putative N-glycosylation consensus sequence Asn-X-Thr/Ser. This mutation could alter the polysaccharide content of Kell and lead to a change in protein conformation and/or trafficking into the cellular machinery (29), which could in turn affect its function.…”

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confidence: 99%

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The Kell Protein of the Common K2 Phenotype Is a Catalytically Active Metalloprotease, whereas the Rare Kell K1 Antigen Is Inactive

Clapéron

Rose

Gane

et al. 2005

Journal of Biological Chemistry

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The Kell blood group is a highly polymorphic system containing over 20 different antigens borne by the protein Kell, a 93-kDa type II glycoprotein that displays high sequence homology with members of the M13 family of zinc-dependent metalloproteases whose prototypical member is neprilysin. Kell K1 is an antigen expressed in 9% of the Caucasian population, characterized by a point mutation (T193M) of the Kell K2 antigen, and located within a putative N-glycosylation consensus sequence. Recently, a recombinant, non-physiological, soluble form of Kell was shown to cleave Big ET-3 to produce the mature vasoconstrictive peptide. To better characterize the enzymatic activity of the Kell protein and the possible differences introduced by antigenic point mutations affecting post-translational processing, the membrane-bound forms of the Kell K1 and Kell K2 antigens were expressed either in K562 cells, an erythroid cell line, or in HEK293 cells, a non-erythroid system, and their pharmacological profiles and enzymatic specificities toward synthetic and natural peptides were evaluated. Results presented herein reveal that the two antigens possess considerable differences in their enzymatic activities, although not in their trafficking pattern. Indeed, although both antigens are expressed at the cell surface, Kell K1 protein is shown to be inactive, whereas the Kell K2 antigen binds neprilysin inhibitory compounds such as phosphoramidon and thiorphan with high affinity, cleaves the precursors of the endothelin peptides, and inactivates members of the tachykinin family with enzymatic properties resembling those of other members of the M13 family of metalloproteases to which it belongs.The Kell blood group is a highly polymorphic system containing over 20 different antigens, many of which are organized in five antithetical sets with opposing high and low incidence while others remain independently expressed. One of the best characterized such set of antigens is also the first to be identified and is composed of the low prevalence K1 1 antigen (K, Kell), expressed in 9% of the Caucasian population, linked to maternal alloimmunization and hemolytic disease of the newborn. Its antithetical form is K2 (k, cellano), and it is expressed in over 90% of the population (1, 2). The complex Kell blood group was long known as carried by a single gene (KEL) coding for a 93-kDa surface-exposed glycoprotein expressed on red cell membranes where the Kell protein has been shown to be covalently linked to a polytopic membrane protein of 37 kDa, Kx. The absence of Kx leads to a pathological condition known as McLeod syndrome, which associates to red cell acanthocytosis as well as muscular and neurological disorders (3, 4). The molecular cloning of the gene coding the Kell protein (5) has opened new paths in the study of the Kell blood group system as the molecular basis of the various antigenic forms were brought to light and with the revelation of its high degree of sequence homology with the members of the M13 family of zinc-dependent metallop...

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Section: Discussionsupporting

confidence: 94%

mentioning

confidence: 99%

The Kell Protein of the Common K2 Phenotype Is a Catalytically Active Metalloprotease, whereas the Rare Kell K1 Antigen Is Inactive

Clapéron

Rose

Gane

et al. 2005

Journal of Biological Chemistry

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“…41 Because KEL6 has an incidence of 20% in persons of African heritage and occurs only in about 0.01% in whites, it is thought that this unequal distribution may reflect a selection pressure that influences the functional properties of Kell. However, the model does not indicate that the Leu597Pro mutation is in close spatial relation with the enzymeactive site.…”

Section: Kell Blood Group Antigens Are Clustered On the Nonconserved mentioning

confidence: 99%

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Lee

Debnath

Redman

2003

Blood

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In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3-converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of ␣-helical segments. The domain situated closest to the membrane contains both the N-and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. IntroductionThe Kell blood group protein is a type II membrane glycoprotein that is important in transfusion medicine due to the immunogenicity of its many polymorphic forms. The primary structure of Kell protein and its enzyme function as an endothelin-converting enzyme, classifies it as a member of the neprilysin (M13) subfamily of zinc endopeptidases. Kell differs from other M13 members in that it is linked by a single disulfide bond to another protein, XK, which traverses the membrane 10 times (for reviews, see Lee et al 1,2 ). XK has the structural characteristics of a membrane transporter, but its function has not yet been determined. 3 As a group the mammalian M13 peptidases are involved in the activation of bioactive peptides by specific cleavages of larger inactive peptides and in the proteolysis of bioactive peptides. [4][5][6] The substrate specificities of the M13 family members vary with neutral endopeptidase (NEP; EC 3.4.24.11) 7 and secreted endopeptidase (SEP) 8 having a wide range of substrates and endothelinconverting enzyme 1 (ECE-1; EC 3.4.24.71), endothelin-converting enzyme 2 (ECE-2), 9,10 Kell, 11 and phosphate-regulating protein (PHEX) 12,13 with a restricted number of substrates. ECE-1 and ECE-2 preferentially cleave big endothelin-1-releasing endothelin-1 (ET-1) but can also cleave big endothelin-2 and big endothelin-3. 14 ECE-1 also hydrolyzes other bioactive peptides such as bradykinin, neurotensin, and substance P. 9 Kell, like ECE-1 and ECE-2, is also an endothelin-converting enzyme but, in contrast to ECE-1 and ECE-2, Kell preferentially cleaves big endothelin-3, releasing the active peptide endothelin-3 (ET-3) although it also, to a lesser degree, activates ET-1 and endothelin-2 (ET-2). 11 Thus far the parathyroid hormone-related fragment 107-139 and fibroblast growth factor 23 (FGF-23), which is involved in phosphate transport in the kidney, are the only known substrates for PHEX. 12,13 Substrates for XCE, which is mostly expressed in the central nervous system, have not yet been identified. 15,16 Site-directed mutagenesis of conserved amino acid residues in NEP have yielded valuab...

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“…For example, KEL1 is present in 9% of Caucasians and in 2% of persons of African heritage, whereas KEL6 is present in 20% of persons of African heritage and in less than 1% of Caucasians. KEL1 is due to a 698C3 T mutation that results in a Thr 193 3 Met substitution at an N-glycosylation consensus sequence (3). KEL6 is due to a 1910T3 C mutation that results in a Leu 597 3 Pro substitution, which is located between two closely placed non-conserved cysteine residues (596CPAC599), and the antigen is more sensitive to reducing agents (4).…”

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confidence: 99%

Endothelin-3-converting Enzyme Activity of the KEL1 and KEL6 Phenotypes of the Kell Blood Group System

Sha

Redman

Lee

2006

Journal of Biological Chemistry

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The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp 21 -Ile 22 bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype. We demonstrate that KEL1 red blood cells and also a soluble recombinant form of KEL1 protein (s-Kell KEL1) have similar enzymatic activity as the common Kell phenotype. In addition we show that KEL6 red blood cells, which are more prevalent in persons of African heritage than in Caucasians also have endothelin-3-converting enzyme activity and that the recombinant soluble form of KEL6 protein (s-Kell KEL6) has similar K m values as the wild-type.The Kell blood group system is highly polymorphic, expressing over 25 antigens that have been classified into five antithetical sets of high and low prevalence antigens with the others being independently expressed or having unknown antithetical partners. The different phenotypes are due to single nucleotide mutations that result in an amino acid substitution (1, 2). Two antithetical allelic sets of antigens, KEL1 (K, K1)/KEL2 (k, K2) and KEL6 (Js a )/KEL7(Js b ) are of particular interest because they are more prevalent in certain racial groups. For example, KEL1 is present in 9% of Caucasians and in 2% of persons of African heritage, whereas KEL6 is present in 20% of persons of African heritage and in less than 1% of Caucasians. KEL1 is due to a 698C3 T mutation that results in a Thr 193 3 Met substitution at an N-glycosylation consensus sequence (3). KEL6 is due to a 1910T3 C mutation that results in a Leu 597 3 Pro substitution, which is located between two closely placed non-conserved cysteine residues (596CPAC599), and the antigen is more sensitive to reducing agents (4).The Kell blood group protein, a 93-kDa, type II membrane glycoprotein, is a member of the M13 family of zinc endopeptidases (5). Within this large group, Kell has significant similarity with six other type II membrane glycoproteins: neutral endopeptidase 24.11 (6), neprilysin 2 (NL1/SEP) (7-9), two different endothelin-converting enzymes (ECE-1 and ECE-2) (10, 11), PEX (PHEX) (12), and X-converting enzyme (endothelin-converting enzyme-like 1/damage-induced neutral endopeptidase; ECEL1/DINE) (13). As a group, the M13 family of zinc endopeptidases either degrades bioactive peptides or cleaves intermediate inactive peptides to produce smaller bioactive peptides (14). However, in some cases, for example X-converting enzyme, the substrates are unknown. Unlike endothelin-converting enzyme-1 and -2 that preferentially activate endothelin-1 (ET 2 -1) (10, 11), Kell has strong preference for big endothelin-3 (big ET-3) and has much less activity with big endothelin-1 (big ET-1) and big endothelin-2 (bi...

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Molecular basis of the K:6,‐7 [Js(a+b−)] phenotype in the Kell blood group system

Abstract: The base substitutions T-to-C at nt 1910 and A-to-G at nt 2019 are unique to KEL6. The predicted Leu-->Pro change may disrupt the alpha-helical structure and thus form the epitope for KEL6.

Cited by 46 publications

References 14 publications

The Kell Protein of the Common K2 Phenotype Is a Catalytically Active Metalloprotease, whereas the Rare Kell K1 Antigen Is Inactive

The Kell Protein of the Common K2 Phenotype Is a Catalytically Active Metalloprotease, whereas the Rare Kell K1 Antigen Is Inactive

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Endothelin-3-converting Enzyme Activity of the KEL1 and KEL6 Phenotypes of the Kell Blood Group System

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