The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.
Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Yi-Chieh
YC
Lin
Bing-Mae
BM
Chen
Wei-Cheng
WC
Lu
Chien-I
CI
Su
Zeljko M
ZM
Prijovich
Wen-Chuan
WC
Chung
Pei-Yu
PY
Wu
Kai-Chuan
KC
Chen
I-Chiao
IC
Lee
Ting-Yi
TY
Juan
Steve R
SR
Roffler
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018122:B7-1 Antigen / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000378:metabolism
D018122:B7-1 Antigen; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins