Functional expression of the chicken low density lipoprotein receptor-related protein in a mutant chinese hamster ovary cell line restores toxicity of Pseudomonas exotoxin A and degradation of alpha2-macroglobulin.
The low density lipoprotein receptor-related protein (LRP) is responsible for the clearance of several physiological ligands including a complex of proteinase and alpha2-macroglobulin (alpha2M) and for the entrance of Pseudomonas exotoxin A (PEA) into cells. We have prepared expression plasmids for the full-length chicken LRP (designated LRP100) and two intermediates encoding 25 and 67% of the receptor (designated LRP25 and LRP67, respectively) using overlapping cDNA fragments. LRP25 and LRP67 encode the N-terminal 22 and 64%, respectively, of LRP100 plus the transmembrane and intracellular domains. Transient transfection of these plasmids into COS-7 cells yielded recombinant proteins of expected molecular mass and immunoreactivity. However, LRP100 was incompletely processed into alpha- (515-kDa) and beta- (85-kDa) chains and was poorly transported from the endoplasmic reticulum to the Golgi compartment. Stable transformants of LRP100, LRP67, and LRP25 were generated in a mutant Chinese hamster ovary cell line that lacked expression of endogenous LRP and was resistant to PEA. All forms of recombinant LRP proteins were transported from the endoplasmic reticulum to the Golgi apparatus in Chinese hamster ovary cells as shown by their sensitivity to endoglycosidase H and resistance to neuraminidase. Cell surface iodination and subcellular fractionation studies indicated that all three LRP variants were expressed on the plasma membrane. Furthermore, expression of the three LRP variants restored, to various degrees, sensitivity to PEA and the ability to degrade methylamine-activated alpha2M (alpha2M*). These data suggest that deletion of large internal portions of LRP, including the processing site, does not prevent transport of LRP to the plasma membrane, nor does it abolish the interaction of LRP with alpha2M* or PEA. This LRP expression system may allow for the characterization of domains within LRP responsible for its multifunctionality.
Avramoglu
R K
RK
Lipoprotein & Atherosclerosis Group, Department of Pathology, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4E9, Canada.
Nimpf
J
J
McLeod
R S
RS
Ko
K W
KW
Wang
Y
Y
FitzGerald
D
D
Yao
Z
Z
D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D016466:CHO Cells; D002645:Chickens; D006224:Cricetinae; D005098:Exotoxins / Q000633:toxicity*; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D009154:Mutation; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014162:Transfection; D037521:Virulence Factors; D000511:alpha-Macroglobulins / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D037521:Virulence Factors; D000511:alpha-Macroglobulins; D000097668:Pseudomonas aeruginosa Exotoxin A