Abstract
We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.
Highlights
Uniuersity School of Medicine, R420, Boston, Massachusetts 02118and the I(CardiovascularResearch Institute, University of California MedicalCenter, Sun Francisco, California94143
Have generated cell lines which express normal and the interaction of several domains of apoA-I are remutant apoA-I forms.Point mutations were introducedquired for the activatioonf LCAT
Fragment 1 was digested with SacII and partially with BssH2, and fragment 2was digested with BssHSIXbaI (Fig. 1).The two fragments were cloned simultaneously into SacIIIXbaI sites of the PUCA-IN vector to generate the A-I(A148-186) mutant
Summary
Single Base Substitutions-Single base substitutions in the apoAI gene were generated by oligonucleotide single priming site-directed mutagenesis [19]. Fromthe replicative form of theresulting m13mp18am4AI(Gln-*,Gln" --f Arg,Arg), the 0.7-kb BglII-Sac fragment was isolated and used to replace the corresponding wild type sequence of the plasmid PUC19A-I This plasmid contains the2.2-kilobase pair PstlPstI apoA-I gene fragment which encompasses theentire coding region and 270 nucleotides upstream of the ATG and 905 nucleotides downstream of the poly(A) site [24, 25]. Fragment 1 was digested with SacII and partially with BssH2, and fragment 2was digested with BssHSIXbaI (Fig. 1).The two fragments were cloned simultaneously into SacIIIXbaI sites of the PUCA-IN vector to generate the A-I(A148-186) mutant This mutant contains three additional amino acids, I, S, and S, between residues 148 and 187 (Fig. 1). To generate media for use in functional assays (see below), transfected cells plated in 150-mm dishes were rinsed three times with phosphate-buffered saline and preincubated twicefor 2 h in SFDMEM containing 10 p~ CdC12.Cells werethen incubated overnight in serum-free DMEM.
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