title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.
abstract: LC3 is widely used marker for macroautophagy assays. After translation pro-LC3 is processed by Atg4 to expose C-terminal glycine residue for downstream conjugation reactions to accomplish the conversion of LC3-I to LC3-II. SDS-PAGE based Western blot (Wb) is generally utilized to quantify LC3-II levels where the LC3-I band migrates slower than LC3-II. We found that pro-human LC3B migrated at similar rate as LC3B-II in SDS-PAGE. The carboxyl-terminal five amino acids, particularly Lysine(122) and Leucine(123) of human LC3B play a major role in the faster migration of unprocessed LC3B, rendering it indistinguishable from LC3B-II in Wb assays. The unique faster migration of unprocessed LC3B than LC3B-I is also revealed in mouse LC3B, rat LC3B and rat LC3 but not in human LC3C. Our findings for the first time define pro-LC3 migration patterns for LC3 family member from human, mouse and rat species in SDS-PAGE. These findings provide a reference for pro-LC3 band patterns when Atg4 function is inhibited.
authors.affiliation: College of Animal Science and Veterinary Medicine, Jilin University, Jilin, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Zhixia
authors.initials: Z
authors.lastname: Chen
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.forename: Michael T
authors.initials: MT
authors.lastname: Stang
authors.forename: Wentao
authors.initials: W
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy / Q000235:genetics*; D000071183:Autophagy-Related Proteins; D015153:Blotting, Western; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D013045:Species Specificity
chemical_list: D000071183:Autophagy-Related Proteins; D008055:Lipids; C477012:MAP1LC3A protein, human; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D011498:Protein Precursors; C474025:ATG4A protein, human; D003546:Cysteine Endopeptidases; D007930:Leucine; D008239:Lysine
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