High transcytosis of melanotransferrin (P97) across the blood–brain barrier

Abstract

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration-and conformationdependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Directing adenovirus across the blood–brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model

“…This temperature dependence was demonstrated for recombinant MTf transcytosis. 11 To confirm Ad5 vectors were indeed transported across the endothelial barrier via transcytosis pathway, we examined temperature dependence of this process by performing the transcytosis assay at either 4 or 371C. As shown in Figure 4a, sCAR-MTf-mediated transcytosis was completely inhibited at 41C (Po0.01), whereas in the control experiment with sCAR, there was no significant difference between 4 and 371C (P40.05).…”

“…As previous studies have shown that MTf prefers apical-to-basal transcellular transport, 11 in the transcytosis assay, we pre-mixed Ad5 VPs with sCAR-MTf or control sCAR, and added the complexes to the upper chamber (apical side of the barrier). The cells were then sCAR-MTf-mediated Ad5 transcytosis across the BBB Y Tang et al incubated for 6 h in a 371C, 5% CO 2 -containing humidified incubator to allow transcytosis to occur.…”

“…[8][9][10] Recent studies discovered that human melanotransferrin (MTf, also named P97), a transferrin homolog originally identified in human melanoma, was highly accumulated into the mouse brain following intravenous injection. 11,12 These studies demonstrated that MTf could cross the brain endothelial cells without affecting the integrity of the BBB and with a much higher rate than what is seen with transferrin and albumin. 11,12 These studies suggest that MTf could be a good candidate for drug delivery to brain.…”

“…11,12 These studies demonstrated that MTf could cross the brain endothelial cells without affecting the integrity of the BBB and with a much higher rate than what is seen with transferrin and albumin. 11,12 These studies suggest that MTf could be a good candidate for drug delivery to brain. It should be noted that these studies were performed with human MTf in mouse brain, and in in vitro BBB models established with bovine brain microvascular endothelial cells (BBMVEC), suggesting MTf functions without significant species discrepancy.…”

“…13 It has been demonstrated that transendothelial transport of MTf occurs via receptor-mediated endocytosis, and low-density lipoprotein (LDL) receptor-related protein (LRP) appears to be involved in MTf transendothelial transport. 11 In this study, we attempted to re-direct Ad5 vectors to the MTf transcytosis pathway so that they can deliver transgenes across the BBB in an in vitro model system. Infection of Ad5 is initiated by attachment of its capsid protein fiber to the cell surface coxsackie-adenovirus receptor (sCAR), followed by interaction of another capsid protein, penton base, with cell surface a v integrins that triggers the internalization of the viruses.…”