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.
Highlights
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
Hematopoietic and Endothelial Cells—Kell K1 and Kell K2 antigens were expressed in two types of human cell lines: K562 cells, which are hematopoietic malignant cells expressing endogenous Kell, and HEK293 cells, kidney fetal cells devoid of
The transfection of Kell K1 and Kell K2 constructs in K562 and HEK293 cells resulted in the expression of a protein of ϳ115 kDa for the K2 antigen and ϳ110 kDa for K1 as revealed by immunoblotting experiments using the
Summary
Materials—Synthetic fluorogenic and bioactive peptides were purchased from Bachem (Weil am Rhein, Germany). A series of synthetic fluorogenic peptides (20 M) were tested using 10 g of total protein from the Kell K1 and Kell K2 lectin purification step (concanavalin A) and diluted in the reaction buffer (50 mM HEPES, pH 7, containing 0.15 M NaCl, 0.01% Triton X-100). For Km determinations, the incubation mixtures were comprised of 10 g of total protein from the lectin fraction of either Kell K1 or K2 HEK293 cell membranes and from 1 to 128 M Glut-FAAF-AMC or Suc-AAF-AMC diluted in 100 l of reaction buffer. For Km determinations, 5 g of protein from the Centricon YM 100 fraction of either Kell K1 or K2 were incubated with increasing concentrations (1.25–160 M) of substrate, i.e. substance P and neurokinin A diluted in 50 l of 50 mM HEPES, pH 7, supplemented with 0.15 M NaCl. All of the samples were analyzed by mass spectrometry coupled to HPLC. Mass spectrometry/mass spectrometry fragmentation data allowed an unambiguous identification as well as quantification of the different substrates and their metabolites using external standards and the LCQuan/Excalibur software
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