The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells.
Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.
Abulrob
Abedelnasser
A
Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
Sprong
Hein
H
Van Bergen en Henegouwen
Paul
P
Stanimirovic
Danica
D
D000584:Amiloride / Q000494:pharmacology; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D001812:Blood-Brain Barrier / Q000502:physiology*; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000166:cytology*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D003847:Deoxyglucose / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods; D006494:Heparin Antagonists / Q000494:pharmacology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D011479:Protamines / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D014316:Tritium / Q000378:metabolism
D000906:Antibodies; D051242:Caveolin 1; D002966:Clathrin; D004791:Enzyme Inhibitors; D006494:Heparin Antagonists; D007143:Immunoglobulin Heavy Chains; D011479:Protamines; D026941:Sodium Channel Blockers; D014316:Tritium; D013395:Sucrose; D000584:Amiloride; D019810:Sodium Azide; D003847:Deoxyglucose; D009439:Neuraminidase