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

Abstract

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...

Identification, immunomodulatory activity, and immunogenicity of the major helper T-cell epitope on the K blood group antigen

“…28 The second characteristic of the K polymorphism that may enhance its ability to induce Th responses is the loss of the glycosylation motif encoded by the antithetical k sequence. 9,31 The Kell protein displays carbohydrate residues at amino acid 191 in the k, but not K, type because substitution of T 193 for M 193 ablates the N-glycosylation site. 9,31 Although this loss does not appear to affect Ab binding, 9,31 there is growing evidence that posttranslational modifications such as glycosylation play an important role in T-cell recognition of Ags.…”

“…9,31 The Kell protein displays carbohydrate residues at amino acid 191 in the k, but not K, type because substitution of T 193 for M 193 ablates the N-glycosylation site. 9,31 Although this loss does not appear to affect Ab binding, 9,31 there is growing evidence that posttranslational modifications such as glycosylation play an important role in T-cell recognition of Ags. 35 There was a relatively high background of Th responses to self-k peptides among alloimmunized donors and unimmunized control volunteers when these peptides were synthesized by standard chemistry with no glycosylation, indicating a lack of tolerance to the primary amino acid sequence.…”

Neurodegeneration in the elderly – When the blood type matters: An overview of the McLeod syndrome with focus on hematological features

“…The Kell glycoprotein (CD238) is a type II single-pass transmembrane red blood cell protein containing 732 aminoacids and functions as an endopeptidase which cleaves big-endothelin3 into the active endothelin3, that acts as a potent vasoconstrictor 16,17 . It expresses at least 35 recognized blood group antigens, including 5 antithetical pairs exerting clinical relevance 18 .…”

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

“…The 230-bp cDNA segment with the KEL1 mutation, obtained by cutting the PCR product with EcoRI and AvrII, replaced a DNA segment between EcoRI and AvrII sites of a wild-type s-Kell construct with an N-terminal six-histidine tag, which was in a pAcGp67A vector (BD Biosciences) (15,20). The sequence of the construct was verified by DNA sequencing.…”

“…The primer sequences of the two PCRs are as follows: PCR1, (EX1F) 5Ј-CAGTCC-TCCGAATCAGCTCCTAGA-3Ј and (K1R) 5Ј-ACTGACTCATCAG-AAGTCTCAGCATTCGG-3Ј; PCR2, (NK1F) 5Ј-GGACTTCCTTAA-ACTTTAACCGAATGCTG-3Ј and (2316R) 5Ј-GCTGTGGCATCT-TTGGTACC-3Ј. The underlined bold letter in the primer sequence is the incorporated KEL1 mutation.The 230-bp cDNA segment with the KEL1 mutation, obtained by cutting the PCR product with EcoRI and AvrII, replaced a DNA segment between EcoRI and AvrII sites of a wild-type s-Kell construct with an N-terminal six-histidine tag, which was in a pAcGp67A vector (BD Biosciences) (15,20). The sequence of the construct was verified by DNA sequencing.…”