Homophilic anchorage of brain-hexokinase to mitochondria-porins revealed by specific-peptide antibody cross recognition.
In brain tumors the main source of energy is from glycolysis, which is initiated by hexokinase 1 (HK1), an enzyme bound to the mitochondrial porin. Disruption of HK binding greatly affects tumor cell survival. Little is known about the acceptor site of HK1. Therefore, a polyclonal antibody (Pab) directed to MIAAQLLAYYFTELK (MK) peptide, corresponding to the 15-amino acids of the N-terminal sequence of brain HK1 was obtained. Anti MK antibody (aMK-Pab)bound specifically to HK as shown by ELISA. The aMK-Pab binding to MK peptide was antibody-concentration dependent and was completely abolished by its preincubation with the peptide at 6 x 10-8 M. The aMK-Pab recognized cytosolic HK (cHK) and HK solubilized (sHK)from rat-brain mitochondrial preparations, but not the yeast HK which does not have the MK sequence. An anti-brain HK Pab (aHK-Pab) directed to purified HK recognized the MK peptide; aHK-Pab bound to MK and this binding was inhibited by preincubation of the antibody with the MK peptide. It was previously demonstrated that brain HK anchors to mitochondria porins, also designated as voltage dependent-anion channels (VDAC) via the MK sequence. A specific anti-VDAC antibody (aVDAC-Pab) which specifically bound the N and C-terminal sequences of VDACwas found to bind to c-HK, sHK and MK-coated wells and this binding was abolished by aVDACPabpreincubation with MK peptide. These data suggest that the three Pabs cross-react with an epitope present in HK and VDAC, and which was presented in the MK peptide. Comparison of alignment of HK or VDAC sequences, available in the protein data bank (PDB), did not allow putative homologues responsible for the cross-reaction to be identified, suggesting that the epitope is conformational. This, added to inhibition of mitochondria-isolated HK binding by the MK peptide,suggests that there is an homophilic-type interaction between HK and porin, through a peptidic structure represented at least in part in the MK peptide.
Oudard
Stéphane
S
CNRS UMR 147, Institut Curie, 75248 Paris Cedex 05, France.
Miccoli
Laurent
L
Beurdeley-Thomas
Arnaud
A
Dutrillaux
Bernard
B
Poupon
Marìe-France
MF
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D003429:Cross Reactions; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006019:Glycolysis; D006593:Hexokinase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D018272:Porins / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D050994:Voltage-Dependent Anion Channels
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D018272:Porins; D050994:Voltage-Dependent Anion Channels; D004024:Dicyclohexylcarbodiimide; C472074:HK1 protein, human; D006593:Hexokinase