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BackgroundBrain lipoprotein metabolism is dependent on lipoprotein particles that resemble plasma high‐density lipoproteins but that contain apolipoprotein (apo) E rather than apoA‐I as their primary protein component. Astrocytes and microglia secrete apoE but not apoA‐I; however, apoA‐I is detectable in both cerebrospinal fluid and brain tissue lysates. The route by which plasma apoA‐I enters the central nervous system is unknown.Methods and ResultsSteady‐state levels of murine apoA‐I in cerebrospinal fluid and interstitial fluid are 0.664 and 0.120 μg/mL, respectively, whereas brain tissue apoA‐I is ≈10% to 15% of its levels in liver. Recombinant, fluorescently tagged human apoA‐I injected intravenously into mice localizes to the choroid plexus within 30 minutes and accumulates in a saturable, dose‐dependent manner in the brain. Recombinant, fluorescently tagged human apoA‐I accumulates in the brain for 2 hours, after which it is eliminated with a half‐life of 10.3 hours. In vitro, human apoA‐I is specifically bound, internalized, and transported across confluent monolayers of primary human choroid plexus epithelial cells and brain microvascular endothelial cells.ConclusionsFollowing intravenous injection, recombinant human apoA‐I rapidly localizes predominantly to the choroid plexus. Because apoA‐I mRNA is undetectable in murine brain, our results suggest that plasma apoA‐I, which is secreted from the liver and intestine, gains access to the central nervous system primarily by crossing the blood–cerebrospinal fluid barrier via specific cellular mediated transport, although transport across the blood–brain barrier may also contribute to a lesser extent.

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

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The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of Type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors.