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 45 kDa 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.