ISOFORM-I (MDR3) IS THE MAJOR FORM OF P-GLYCOPROTEIN EXPRESSED IN MOUSE-BRAIN CAPILLARIES - EVIDENCE FOR CROSS-REACTIVITY OF ANTIBODY-C219 WITH AN UNRELATED PROTEIN
L. Jette et al., ISOFORM-I (MDR3) IS THE MAJOR FORM OF P-GLYCOPROTEIN EXPRESSED IN MOUSE-BRAIN CAPILLARIES - EVIDENCE FOR CROSS-REACTIVITY OF ANTIBODY-C219 WITH AN UNRELATED PROTEIN, Biochemical journal, 305, 1995, pp. 761-766
P-glycoprotein (P-gp) is expressed in various non-cancerous tissues su
ch as the endothelial cells of the blood-brain barrier. We used severa
l monoclonal antibodies (mAbs) and isoform-specific polyclonal antibod
ies to establish which P-gp isoforms are expressed in isolated mouse b
rain capillaries. P-gp class I isoform was detected in capillaries wit
h a Western immunoblotting procedure using a specific antiserum. No im
munoreactivity was observed with either class II- or class III-specifi
c antisera, Immunoreactivity was observed with mAb C219. However, this
antibody detected two distinct immunoreactive proteins (155 and 190 k
Da) in the isolated brain capillaries. These two proteins comigrated a
s a broad band when the samples were submitted to heat prior to gel el
ectrophoresis. The glycoprotein nature of these two antigens was evalu
ated by their sensitivity to N-glycanase treatment. Following this tre
atment, the size of the proteins was reduced from 190 and 155 kDa to 1
80 and 120 kDa, respectively. Triton X-114 phase-partitioning studies
showed that the 190 kDa immunoreactive protein was poorly solubilized
by Triton X-114, while the 155 kDa protein was partitioned in the dete
rgent-rich phase. In labelling experiments, only the 155 kDa protein w
as photolabelled with [I-125]iodoarylazidoprazosin. These results show
that a 190 kDa protein detected by antibody C219 is an antigen unrela
ted to the three P-gp isoforms presently known. Cross-reactivity of C2
19 with an unrelated protein emphasizes the fact that more than one an
tibody should be used in the assessment of P-gp expression in cell lin
es and tissues.