title: A basic phenylalanine-rich oligo-peptide causes antibody cross-reactivity.
abstract: Glycolate oxidase (GO) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) are the two enzymes that serve key functions in the photorespiration and photosynthesis of plants. A 2 kDa highly basic phenylalanine-rich oligo-peptide (BOP) binds to the surface of acidic GO via ionic and hydrophobic interactions, forming the GO-BOP complex (GC). Previously, RubisCO was thought to exist as a single species composed of a large (rbc L, 54 kDa) and a small subunit (rbc S, 14 kDa). Here we show for the first time, using 2-DE, SDS-PAGE, immunoassays and amino acid determination, that BOP also interacts with RubisCO and that many RubisCO-BOP complexes (RCs), differing in pI, hydrophobicity and activity, coexist in green leaves. GCs, RCs and crude extract from green leaves analyzed by SDS-PAGE Western blotting showed that BOP exists either in subunit-BOP complexes (GO subunit-BOP, rbc L-BOP and rbc S-BOP etc.), with a wide variation in the number and the position of BOPs bound to each subunit molecular, or alone without a binding partner. When rbc L-BOP and rbc S-BOP were assayed by SDS-PAGE, BOP was dissociated from the subunit and it self-assembled to form 37 different BOP polymers (basic phenylalanine-rich protein) whose molecular weights (M(r)s) ranged from 34.0 to 91.6 kDa, indicating that the M(r) of BOP is about 2 kDa. Thus, the addition of BOP changes the M(r) of the subunit-BOP complexes so minimally that the rbc L and rbc S run at their predicted M(r)s on SDS-PAGE. In summary, the results described here demonstrate that the presence of BOP in complexes (both subunit-BOP complex and protein-BOP complex) can cause cross-reactivity of antibodies against different proteins.
authors.affiliation: Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou, P. R. China.
authors.forename: Gangyue
authors.initials: G
authors.lastname: Luo
authors.forename: Guanghua
authors.initials: G
authors.lastname: Quan
authors.forename: Jinya
authors.initials: J
authors.lastname: Guo
authors.forename: Haijiang
authors.initials: H
authors.lastname: Zhang
authors.forename: Sun
authors.initials: S
authors.lastname: Li
authors.forename: Weidong
authors.initials: W
authors.lastname: Wu
authors.forename: Ling
authors.initials: L
authors.lastname: Nie
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Dong
authors.forename: Suhong
authors.initials: S
authors.lastname: Wu
authors.forename: Guangni
authors.initials: G
authors.lastname: Zheng
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.forename: Weina
authors.initials: W
authors.lastname: Wang
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D001937:Brassica / Q000737:chemistry; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D007118:Immunoassay; D051379:Mice; D008970:Molecular Weight; D046912:Multiprotein Complexes; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D016133:Polymerase Chain Reaction; D021122:Protein Subunits; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000906:Antibodies; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010936:Plant Extracts; D021122:Protein Subunits; D010649:Phenylalanine; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase; D012273:Ribulose-Bisphosphate Carboxylase