Proteolytic Processing of Big Endothelin-3 by the Kell Blood Group Protein

Abstract

Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.

McLeod phenotype associated with a XK missense mutation without hematologic, neuromuscular, or cerebral involvement

“…The Kell protein is an endothelin-3 converting enzyme generating the bioactive endothelin-3. 14,33 Experimental studies demonstrated that endothelin is a neurotrophic factor at low concentrations and a cytotoxic factor at high concentrations, suggesting that endothelinrelated mechanisms might be implicated in neurodegeneration. 34,35 However, no acanthocytosis, cerebral, or neuromuscular signs and symptoms have been described in individuals harboring the K 0 -phenotype.…”

“…9,11,12 Although the precise function of the XK/Kell-complex is not yet clarified, available data suggest an important role in muscle and striatal nerve cell physiology. [12][13][14] The vast majority of disease-causing XK mutations comprise deletions, nonsense mutations, or splicesite mutations predicting absent or truncated XK protein devoid of the Kell protein binding site. 3,4,9,15,16 To date, only two XK missense mutations have been described.…”

Blood groups: the past 50 years

“…31 The Kell blood group antigen is an active enzyme: an endopeptidase that processes endothelin-3, a potent vasoconstrictor, by cleavage of its biologically inactive progenitor. 32 Its function in vivo, however, remains unclear and it is not known whether the Kell glycoprotein processes any other biopeptides. No obvious pathology is associated with the Kell-null phenotype.…”

The value of DNA analysis for antigens of the Kell and Kx blood group systems

“…1,2 Kell, an endothelin-3-converting enzyme, is a 93-kDa type II membrane glycoprotein that belongs to the M13 family of zinc endopeptidases. 3,4 Like other members of the M13 family, Kell has an intracellular N-terminal domain and a large extracellular C-terminal domain which contains a zinc-binding catalytic pentapeptide consensus sequence, HExxH (in Kell, HELLH). Kell contains five possible N-glycosylation sites and 15 cysteine residues on the ectodomain, predicting a highly folded nature.…”