ABCMdb

PMID: 11880368

Ilias A, Urban Z, Seidl TL, Le Saux O, Sinko E, Boyd CD, Sarkadi B, Varadi A
Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).
J Biol Chem. 2002 May 10;277(19):16860-7. Epub 2002 Mar 5., 2002-05-10 [PubMed]

Sentences

No. Mutations Sentence Comment

47 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe To study the effect of PXE mutations on MRP6/ABCC6 function, we have inserted three naturally occurring PXE mutations, V1298F, G1302R, and G1321S (20), into our cDNA by using inverse PCR mutagenesis (21). Login to comment 48 ABCC6 p.Gly1302Arg One of the missense mutations (G1302R) we studied was found in homozygous form (20) in two unrelated patients, whereas the other two were detected in heterozygous form. Login to comment 50 ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe The V1298F were present on 2 alleles of the 101 unrelated patients with PXE, whereas the G1321S was present on 1 allele (20). Login to comment 77 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe Using this cDNA with a verified sequence, we have generated recombinant baculoviruses containing the wild type human ABCC6 cDNA as well as that of three ABCC6 mutant variants (V1298F, G1302R, and G1321S). Login to comment 84 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe Moreover, as shown by immunostaining in Fig. 1B, all three mutant forms of human ABCC6 (V1298F, G1302R, and G1321S) were expressed in about the same amount as the wild type protein (94, 78, and 88% of the wild type, respectively, as measured by densitometry) and all with the correct molecular mass. Login to comment 157 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe Transport Studies of Mutants ABCC6 Variants Causing PXE-Based on these results and to analyze the transport activity of the PXE-causing ABCC6 mutations, we studied the NEM-GS and the LTC4 uptake in Sf9 cell membrane vesicles containing three ABCC6 variants, V1298F, G1302R, and G1321S. Login to comment 163 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe Isolated membranes of Sf9 cells expressing wild type or mutant (V1298F, G1302R, and G1321S) ABCC6 transporters were incubated in the presence of 2 ␮M Mg-8-N3-[␥-32 P]ATP and either orthovanadate (1 mM) or AlF4 (2 mM) was added as a trapping anion. Login to comment 173 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe Effect of V1298F, G1302R, and G1321S mutations on the transport and nucleotide-trapping activity of human ABCC6. Login to comment 181 ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe Two of the transport-negative PXE mutants (V1298F and G1321S) could not be labeled under these nucleotide-trapping conditions (Fig. 4C) if either vanadate or AlF4 was present as a complex-stabilizing anion. Login to comment 182 ABCC6 p.Gly1302Arg The G1302R mutant, however, could form the occluded nucleotide intermediate state, as incorporation of radioactive label was detected under catalytic conditions in this mutant (Fig. 4C). Login to comment 223 ABCC6 p.Gly1321Ser ABCC6 p.Gly1302Arg ABCC6 p.Val1298Phe In the present study we expressed and investigated three PXE mutants, V1298F, G1302R, and G1321S, that were all within the C-proximal ABC domain (see Fig. 1A). Login to comment 236 ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe In the present study we found that neither V1298F nor G1321S ABCC6 could be labeled with 8-azido-[␥-32 P]ATP in the presence of vanadate or AlF4 under nucleotide-trapping conditions (see Fig. 4C). Login to comment 239 ABCC6 p.Gly1302Arg Interestingly, nucleotide trapping was performed by the G1302R mutant (Fig. 4C), which suggests that the transport/ ATPase cycle is arrested at a later step in this transport-negative mutant. Login to comment