Expression of monocarboxylate transporter 4 in human platelets, leukocytes, and tissues assessed by antibodies raised against terminal versus pre-terminal peptides.
We compared antibodies (Abs) raised in rabbits against two non-overlapping peptides, terminal (T) and pre-terminal (PT) of the human monocarboxylate transporter (MCT4) lactate transporter in a variety of human tissues. Upon stringent SDS extraction, the PT Ab recognized a major 32 kDa band in many tissues, but not in leukocytes, while the T Ab recognized a 45 kDa band in leukocytes but only in a few other tissues. In two cell lines, human adult retinal pigment epithelial and Madin-Darby canine kidney, however, both Abs identified the same 45 kDa band only, whether extracted by stringent SDS or by a mild Triton X-100 procedure. Applying Triton X-100 and milder SDS methods to human tissues led us to conclude that: (1) MCT4 is more labile to proteolysis than MCT1 or 2; (2) the proteolysis involves an enzyme system which is absent from the cell lines, is of variable content in human tissues, and is accelerated by SDS and/or heat; (3) a major product is the 32 kDa band, which is missing the C-terminal peptide, since it is seen by the Ab to MCT4-PT, but not the Ab to MCT4-T; (4) this truncated 32 kDa form is prone to aggregate, producing oligomers also detected only by the MCT4-PT; (5) the 32 kDa form may have a physiological function, since (except in the cell lines and monocytes) it is the major form seen with the PT Ab even with our mildest extractions, and since MCT4-PT stained two compartments that were not stained by the T Ab in our immunohistochemistry survey: the capsule of the muscle spindle, and the cytoplasm of the lymphocyte; (6) platelets contained MCT4, stained by both Abs, and verified by the 45kDa band on Western blotting, in addition to the presence of MCT2 that we had demonstrated previously [N. Merezhinskaya, S.A. Ogunwuyi, F.G. Mullick, W.N. Fishbein, Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes, J. Histochem. Cytochem. 52 (2004) 1483-1493.
Merezhinskaya
Natalya
N
Environmental and Infectious Disease Sciences Department, Armed Forces Institute of Pathology, Washington, DC, USA. Merezhin@afip.osd.mil
Ogunwuyi
Sunday A
SA
Fishbein
William N
WN
D000818:Animals; D000906:Antibodies; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D004947:Esophagus / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D027501:Monocarboxylic Acid Transporters / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D009132:Muscles / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology*
D000906:Antibodies; D027501:Monocarboxylic Acid Transporters; D009124:Muscle Proteins; D010446:Peptide Fragments; C406962:SLC16A4 protein, human