Purification and Partial Characterization of the Erythrocyte Kx Protein Deficient in McLeod Patients

Khamlichi, Samir; Bailly, Pascal; Blanchard, Dominique; Goossens, D.; Cartron, Jean‐Pierre; Bertrand, Olivier

doi:10.1111/j.1432-1033.1995.tb20342.x

Cited by 75 publications

(44 citation statements)

References 23 publications

(15 reference statements)

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“…Alternatively, conformational changes in D237G and F731Y mutants detected by endoprotease digestion may prevent interaction of the PHEX ectodomain with putative partners at the cell surface. In this regard, it is of interest that KELL, another member of the M13 family of zinc metallopeptidase, has been shown to be associated with a membrane protein XK, which is mutated in patients with McLeod syndrome, a rare X-linked disorder (43,44). The functional role of this association, however, remains to be determined.…”

Section: Discussionmentioning

confidence: 96%

Structure and Function of Disease-Causing Missense Mutations in the PHEX Gene

Sabbagh

Boileau

Campos

et al. 2003

The Journal of Clinical Endocrinology &Amp; Metabolism

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The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.

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Section: Discussionmentioning

confidence: 96%

Structure and Function of Disease-Causing Missense Mutations in the PHEX Gene

Sabbagh

Boileau

Campos

et al. 2003

The Journal of Clinical Endocrinology &Amp; Metabolism

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“…[33,62,69] and cerebral abnormalities demonstrated on cranial imaging [11]. The gene responsible for McLeod syndrome, designated XK, has been isolated in the Xp21 region and found to encode for a novel membrane transport protein [26] that corresponds to the predicted Kx protein [30]. Several separate point mutations have to date been reported in McLeod syndrome [18,26,27,56].…”

Section: Mcleod Syndromementioning

confidence: 98%

Acanthocytosis and neurological disorders

Stevenson

Hardie

2001

Journal of Neurology

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Acanthocytosis occurs because of ultrastructural abnormalities of the erythrocyte membranous skeleton resulting in reduced membrane fluidity. At least three hereditary neurological conditions are associated with it, although as yet the pathogenesis of the neurological features is unknown. In abetalipoproteinaemia, an autosomal recessive condition, vitamin E deficiency results in a progressive spinocerebellar syndrome associated with peripheral neuropathy and retinitis pigmentosa. Neuroacanthocytosis is also probably an autosomal recessive condition and is characterised by chorea, orofaciolingual dyskinesia, dysarthria, areflexia, seizures and dementia. McLeod syndrome is an X-linked recessive disorder usually presenting in males as a benign myopathy with areflexia, in association with a particular abnormality of expression of Kell blood group antigens. However, occasionally the neurological features are more severe and indistinguishable from those of neuroacanthocytosis. Recent advances in molecular genetics may assist better understanding of the disease mechanisms and the search for more effective treatments.

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“…The Kell and Kx proteins are physically associated on the membrane, most likely by disulphide bond(s) (Branch et al, 1985), and this property was used to copurify both proteins with a human monoclonal antibody of Kell blood group specificity (Khamlichi et al, 1995). The N-terminal sequence of the purified Kx protein was found to be identical to the predicted protein product of the XK gene, deficient in the McLeod syndrome, recently isolated by positional cloning (Ho et al, 1994).…”

mentioning

confidence: 99%

Immunochemical analysis of the Kx protein from human red cells of different Kell phenotypes using antibodies raised against synthetic peptides

et al. 1997

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The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription‐translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell–Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including K0, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in K0 cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.

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Purification and Partial Characterization of the Erythrocyte Kx Protein Deficient in McLeod Patients

Cited by 75 publications

References 23 publications

Structure and Function of Disease-Causing Missense Mutations in the PHEX Gene

Structure and Function of Disease-Causing Missense Mutations in the PHEX Gene

Acanthocytosis and neurological disorders

Immunochemical analysis of the Kx protein from human red cells of different Kell phenotypes using antibodies raised against synthetic peptides

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