Identification of Palmitoylated Transitional Endoplasmic Reticulum ATPase by Proteomic Technique and Pan Antipalmitoylation Antibody

Protein palmitoylation plays a significant
role in a wide range
of biological processes such as cell signal transduction, metabolism,
apoptosis, and carcinogenesis. For high-throughput analysis of protein
palmitoylation, approaches based on the acyl-biotin exchange or metabolic
labeling of azide/alkynyl-palmitate analogs are commonly used. No
palmitoylation antibody has been reported. Here, the palmitoylated
proteome of human colon cancer cell lines SW480 was analyzed via a
TS-6B-based method. In total, 151 putative palmitoylated sites on
92 proteins, including 100 novel sites, were identified. Except for
3 known palmitoylated transmembrane proteins, ATP1A1, ZDHHC5, and
PLP2, some important proteins including kinases, ion channels, receptors,
and cytoskeletal proteins were also identified, such as CLIC1, PGK1,
PPIA, FKBP4, exportin-2, etc. More importantly, the pan antipalmitoylation
antibody was developed and verified for the first time. Our homemade
pan antipalmitoylation antiserum could differentiate well protein
palmitoylation from mouse brain membrane fraction and SW480 cells,
which affords a new technique for analyzing protein palmitoylation
by detecting the palmitic acid moiety directly. Furthermore, the candidate
protein transitional endoplasmic reticulum ATPase (VCP) identified
in SW480 cells was validated to be palmitoylated by Western blotting
with anti-VCP antibody and the homemade pan antipalmitoylation antibody.