Establishing biochemical and physiological functions of class 2a putative amphipath transporter
Open Access
- Author:
- Gao, Ling
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 07, 2003
- Committee Members:
- Robert Allen Schlegel, Committee Chair/Co-Chair
Ming Tien, Committee Chair/Co-Chair
James Gregory Ferry, Committee Member
Song Tan, Committee Member
Cooduvalli S Shashikant, Committee Member - Keywords:
- class 2a
Atp9a
baculovirus expression
ATPase
knockout mice - Abstract:
- Phosphatidylserine (PS) is restricted exclusively to the inner leaflet of the bilayer of the plasma membrane of animal cells. This asymmetric distribution is maintained by an aminophospholipid translocase which specifically transports PS from the outer to inner leaflet of the membrane. The gene encoding an aminophospholipid translocase (ATPase II or 1a) has been cloned and found to be a type IV P-type ATPase. In mammals, 14 type IV P-type ATPases have been identified. However, very little is known about them. The goal of this thesis is to elucidate the biochemical and physiological functions of one of these genes, the 2a gene. Using probes generated from a rat 2a EST clone, a mouse brain cDNA library was screened, and overlapping clones containing the entire murine 2a ORF were isolated. The 2a ORF is 1047 aa dispersed in 28 exons, and codes for a protein with a predicted molecular weight of 118.8 kD. To detect expression of the 2a protein, polyclonal antibodies were generated against a 2a-specific peptide of 13 aa, and immunoaffinity-purified using BSA-peptide conjugates coupled to CNBr-activated sepharose beads. The 2a affinity-purified antibody specifically detected proteins of approximately the predicted molecular weight in microsomal fractions of rat PC12 cells and mouse brain. In order to have enough enzyme to investigate what substrate the 2a gene product transports, the 2a ORF was cloned into a baculovirus expression system, containing a putative ribosome binding sequence to enhance expression, and a his-tag at the C-terminus to allow purification. The 120 kD protein expressed was purified to a single band by Ni-NTA column chromatography, and was specifically recognized by both the affinity-purified 2a antibody and a probe detecting his-tagged proteins. To further confirm that the protein at 120 kD is the 2a recombinant protein, the band was excised from the gel, digested with trypsin, and the tryptic peptide mixture analyzed by MALDI mass spectrometry. The observed mass values of 31 peptides matched the masses expected from tryptic digestion of the 2a protein. The sequences of the 31 matched peptides span 36% of the 2a ORF, verifying that the purified protein at 120 kD is the 2a protein. Since only small amounts of the 2a protein could be solubilized from the microsomal fraction by the non-ionic detergent, polyoxyethylene 9 lauryl ether (C12E9), various concentrations of the non-ionic detergents, polyoxyethylene 8 lauryl ether, dodecyl maltoside (DDM), or octyl glucoside were tested for their ability to solubilize the 2a protein. 1% DDM in 4 mg/ml of microsomal protein produced the best solubilization, and solubilized 5 times more 2a protein than 1% C12E9. However, ATPase assays indicated that the 2a enzyme prepared in DDM had much lower specific activity than the enzyme prepared in C12E9. The addition of C12E9 partially restored the activity of the 2a enzyme prepared in DDM, suggesting that DDM might solubilize inactive 2a protein. The ATPase activity of recombinant 1a aminophospholipid translocase is stimulated dramatically by PS. To investigate whether this is also true for the 2a enzyme, the ATPase activity of the 2a eluate purified by Ni-NTA column chromatography was tested in the absence or presence of PS. In the absence of PS, the specific activity of the 2a eluate was 1.8-3 nmol Pi/mg/min. In the presence of 250 uM defined, synthetic PS, the specific activity of the 2a enzyme was 3.1-3.9 nmol Pi/mg/min, indicating that PS stimulated the 2a enzyme activity by only 30-70%. This small stimulation by PS suggests that the 2a enzyme has different biochemical properties from the 1a enzyme, and that PS may not be the substrate of the 2a enzyme. Since all P-type ATPases are vanadate-sensitive, 100 uM vanadate was used to test whether the PS-stimulated ATPase activity of the 2a eluate was vanadate-sensitive. Vanadate inhibited the PS-stimulated activity by 40-70%, suggesting that the activity inhibited may represent that of the 2a enzyme with the remaining activity representing vanadate-insensitive ATPase contaminants. If correct, then testing whether other amphipathic molecules can dramatically stimulate the 2a enzyme activity may identify its substrate. However, it remains possible that the 2a enzyme is misfolded and inactive, and that the vanadate-sensitive activity results from other P-type ATPase contaminants. Since ouabain and EGTA were included in the ATPase assays, that the vanadate-sensitive activity was derived from the Na,K-ATPase or Ca2+-ATPase can be ruled out. To test whether the 2a protein was in a conformation able to bind ATP and thus potentially active if its substrate was present, photoaffinity labeling of the 2a eluates with (a-P32)ATP was performed. Although the protocol used labeled the Na,K-ATPase, another P-type ATPase, the 2a enzyme was not radio-labeled, and neither were any other ATPases which might contaminate the preparation. Thus, at present, it is not known whether the 2a protein is folded properly and can be activated by its substrate, or whether it is not folded properly and the vanadate-sensitive activity measured is that of other contaminating ATPases. To reveal the physiological role of the 2a enzyme, the gene was disabled in mouse embryonic stem (ES) cells in order to generate knockout mice. Genomic clones containing the first four exons of the 2a gene were isolated and sequenced. From these clones, a targeting vector, which contained a neomycin-resistant positive selection cassette replacing exons 3 and 4, a DT-A negative selection cassette to enrich for homologous recombination events, and 5’ and 3’ homologous regions was constructed. The targeting vector was linearized, and electroporated into BK4 and GS1 ES cells. Six homologous recombinant BK4 clones were identified from a total of 206 neomycin-resistant clones by Southern analyses with 5’ and 3’ hybridization probes outside the homologous regions. Six male and three female chimeras with less than 30% agouti coat color were produced from microinjection of homologous recombinant BK4 clones. To generate mice carrying one mutated ES allele, heterozygotes (+/-), the nine chimeras were bred to C57BL/6 mice. Three female and one male chimera were sterile, and a total of 369 pups with black coat color were produced from the other five male chimeras. That only wild-type offspring were obtained indicates that no germline transmission occurred in these chimeras, which might be due to the poor germline transmission ability of the BK4 cell line used at the PSU transgenic facility. Therefore, generation of knockout mice from another ES cell line, J1, was attempted. Eight homologous recombinant J1 clones were identified from 100 neomycin-resistant clones by Southern analyses. One female chimera with 25% agouti coat color and one male chimera with 85% agouti coat color produced from microinjection of the J1 homologous recombinant ES clones are being bred to C57BL/6 mice to generate heterozygotes (+/-). At present, a total of 46 pups with black coat color have been produced from these two chimera. Thus, although I have produced chimeras carrying ES cells in which one 2a allele was mutated, I have not been able to generate germline transmission mice to produce homozygous null mice. Further breeding and microinjection is required.