scholarly journals A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes

2019 ◽  
Vol 32 (5) ◽  
pp. 219-230
Author(s):  
Jason M Lajoie ◽  
Yong Ku Cho ◽  
Dustin Frost ◽  
Samantha Bremner ◽  
Lingjun Li ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Blood Brain Barrier ◽  
Membrane Trafficking ◽  
Plasma Membranes ◽  
Protein Complexes ◽  
Brain Barrier ◽  
Yeast Display ◽  
Functional Protein ◽  
Blood Brain ◽  
Membrane Protein Complexes

Abstract Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

2011 ◽  
Vol 100 (9) ◽  
pp. 3939-3950 ◽  
Author(s):  
Katsuaki Ito ◽  
Yasuo Uchida ◽  
Sumio Ohtsuki ◽  
Sanshiro Aizawa ◽  
Hirotaka Kawakami ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Expression ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Cynomolgus Monkeys ◽  
Membrane Protein Expression ◽  
Blood Brain

Glucose transport by isolated plasma membranes of the bovine blood-brain barrier

1997 ◽  
Vol 272 (5) ◽  
pp. C1552-C1557 ◽  
Author(s):  
W. J. Lee ◽  
D. R. Peterson ◽  
E. J. Sukowski ◽  
R. A. Hawkins
Keyword(s):  
Glucose Transport ◽  
Blood Brain Barrier ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Extracellular Fluid ◽  
Brain Barrier ◽  
Maximal Velocity ◽  
Plasma Membrane Vesicles ◽  
Cerebral Microvessels ◽  
Blood Brain

Luminal and abluminal endothelial plasma membrane vesicles were isolated from bovine cerebral microvessels, the site of the blood-brain barrier. Glucose transport across each membrane was measured using a rapid-filtration technique. Glucose transport into luminal vesicles occurred by a stereospecific energy-independent transporter [Michaelis-Menten constant (K(m)) = 10.3 +/- 2.8 (SE) mM and maximal velocity (Vmax) = 8.6 +/- 2.0 nmol.mg protein(-1).min-1]. Kinetic analysis of abluminal vesicles also showed a transport system with characteristics similar to the luminal transporter (K(m) = 12.5 +/- 2.3 mM and Vmax = 10.0 +/- 1.0 nmol.mg protein-1.min-1). These functional, facilitative glucose transporters were symmetrically distributed between the luminal and abluminal membrane domains, providing a mechanism for glucose movement between blood and brain. The studies also revealed a Na-dependent transporter on the abluminal membrane with a higher affinity and lower capacity than the facilitative transporters (K(m) = 130 +/- 20 microM and Vmax = 1.59 +/- 0.44 nmol.mg protein-1.min-1. The abluminal Na-dependent glucose transporter is in a position to transport glucose from the brain extracellular fluid into the endothelial cells of the blood-brain barrier. The functional significance of its presence there remains to be determined.

scholarly journals Cationic amino acid transport across the blood-brain barrier is mediated exclusively by system y+

2006 ◽  
Vol 291 (2) ◽  
pp. E412-E419 ◽  
Author(s):  
Robyn L. O’Kane ◽  
Juan R. Viña ◽  
Ian Simpson ◽  
Rosa Zaragozá ◽  
Ashwini Mokashi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Amino Acid ◽  
Blood Brain Barrier ◽  
Plasma Membranes ◽  
Plasma Concentrations ◽  
Membrane System ◽  
Brain Barrier ◽  
Cationic Amino Acid ◽  
Blood Brain

Cationic amino acid (CAA) transport is brought about by two families of proteins that are found in various tissues: Cat (CAA transporter), referred to as system y+, and Bat [broad-scope amino acid (AA) transporter], which comprises systems b0,+, B0,+, and y+L. CAA traverse the blood-brain barrier (BBB), but experiments done in vivo have only been able to examine the BBB from the luminal (blood-facing) side. In the present study, plasma membranes isolated from bovine brain microvessels were used to identify and characterize the CAA transporter(s) on both sides of the BBB. From these studies, it was concluded that system y+ was the only transporter present, with a prevalence of activity on the abluminal membrane. System y+ was voltage dependent and had a Km of 470 ± 106 μM (SE) for lysine, a Ki of 34 μM for arginine, and a Ki of 290 μM for ornithine. In the presence of Na+, system y+ was inhibited by several essential neutral AAs. The Ki values were 3–10 times the plasma concentrations, suggesting that system y+ was not as important a point of access for these AAs as system L1. Several small nonessential AAs (serine, glutamine, alanine,and glycine) inhibited system y+ with Ki values similar to their plasma concentrations, suggesting that system y+ may account for the permeability of the BBB to these AAs. System y+ may be important in the provision of arginine for NO synthesis. Real-time PCR and Western blotting techniques established the presence of the three known nitric oxide synthases in cerebral endothelial cells: NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial). These results confirm that system y+ is the only CAA transporter in the BBB and suggest that NO can be produced in brain endothelial cells.

scholarly journals THER-11. PEPTIDE-MEDIATED PERMEABILIZATION OF THE BLOOD-BRAIN BARRIER IMPROVES DRUG DELIVERY TO BRAIN METASTASIS

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i13-i13
Author(s):  
Synnøve Nymark Aasen ◽  
Heidi Espedal ◽  
Christopher Holte ◽  
Olivier Keunen ◽  
Tine Veronika Karlsen ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Blood Brain Barrier ◽  
Plasma Membranes ◽  
Combined Treatment ◽  
Brain Barrier ◽  
Therapeutic Window ◽  
Dce Mri ◽  
Blood Brain ◽  
The Brain ◽  
Bbb Opening

Abstract BACKGROUND: Melanoma patients have a high risk of developing brain metastases, which is associated with a poor prognosis. The blood-brain barrier (BBB) inhibits sufficient drug delivery into metastatic lesions. We investigated the ability of a synthetic peptide (K16ApoE) to permeabilize the BBB for more effective drug treatment. METHODS: DCE-MRI was performed to study the therapeutic window of BBB opening facilitated by K16ApoE. In vivoand in vitroassays were used to determine K16ApoE toxicity and also to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on melanoma metastases was determined together with dabrafenib, which is otherwise known not to cross an intact BBB. RESULTS: DCE-MRI exhibited an effective K16ApoE-mediated BBB opening for up to 1h. Mechanistic studies displayed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At higher concentrations, the peptide also showed unspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice compared to dabrafenib. We also showed by PET/CT that the peptide facilitated the delivery of compounds up to 150 kDa into the brain. CONCLUSIONS: We demonstrate a transient opening of the BBB, caused by K16ApoE, that facilitates improved drug-delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

scholarly journals The Epithelial Membrane Protein 1 is a Novel Tight Junction Protein of the Blood—Brain Barrier

2008 ◽  
Vol 28 (6) ◽  
pp. 1249-1260 ◽  
Author(s):  
Thorsten Bangsow ◽  
Ewa Baumann ◽  
Carmen Bangsow ◽  
Martina H Jaeger ◽  
Bernhard Pelzer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cerebral Ischemia ◽  
Membrane Protein ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
Differentially Expressed ◽  
Brain Barrier ◽  
Brain Vessels ◽  
Blood Brain ◽  
Junction Protein

In the central nervous system, a constant microenvironment required for neuronal cell activity is maintained by the blood—brain barrier (BBB). The BBB is formed by the brain microvascular endothelial cells (BMEC), which are sealed by tight junctions (TJ). To identify genes that are differentially expressed in BMEC compared with peripheral endothelial cells, we constructed a subtractive cDNA library from porcine BMEC (pBMEC) and aortic endothelial cells (AOEC). Screening the library for differentially expressed genes yielded 26 BMEC-specific transcripts, such as solute carrier family 35 member F2 (SLC35F2), ADP-ribosylation factor-like 5B (ARL5B), TSC22 domain family member 1 (TSC22D1), integral membrane protein 2A (ITM2A), and epithelial membrane protein 1 (EMP1). In this study, we show that EMP1 transcript is enriched in pBMEC compared with brain tissue and that EMP1 protein colocalizes with the TJ protein occludin in mouse BMEC by coimmunoprecipitation and in rat brain vessels by immunohistochemistry. Epithelial membrane protein 1 expression was transiently induced in laser-capture microdissected rat brain vessels after a 20-min global cerebral ischemia, in parallel with the loss of occludin immunoreactivity. The study identifies EMP1 as a novel TJ-associated protein of the BBB and suggests its potential role in the regulation of the BBB function in cerebral ischemia.

scholarly journals Antibodies to Blood—Brain Barrier Bind Selectively to Brain Capillary Endothelial Lateral Membranes and to a 46K Protein

1986 ◽  
Vol 6 (2) ◽  
pp. 203-211 ◽  
Author(s):  
William M. Pardridge ◽  
Jing Yang ◽  
Jody Eisenberg ◽  
Lawrence J. Mietus
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Surface Antigen ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Bovine Brain ◽  
Brain Barrier ◽  
Brain Capillary ◽  
Blood Brain ◽  
Capillary Endothelial Cells

To begin elucidating the biochemical basis of the polarized membrane features of the blood–brain barrier (BBB), a series of immunochemical and immunoperoxidase studies were initiated with bovine brain microvessels that make up the BBB in vivo. A rabbit antiserum was prepared against isolated bovine brain BBB plasma membranes. The bovine microvessel plasma membranes were radioiodinated with chloramine-T, and the antiserum selectively immunoprecipitated a 46K protein. The antibodies directed against the 46K protein were quantitatively absorbed with bovine brain capillaries but not with rat kidney or liver powder. Only the capillaries of brain reacted with the rat kidney-absorbed antiserum in immunoperoxidase studies of ethanol-fixed, 8-μm sections of bovine brain cortex, whereas the capillaries in heart, liver, and kidney did not react. This antiserum also strongly illuminated the lateral membranes of isolated bovine brain capillary endothelial cells grown in primary tissue culture. These studies provide evidence for a polarized distribution of a surface antigen in bovine brain capillary endothelial cells that is not present in capillary endothelia of liver, heart, or kidney. The correlation of the immunoperoxidase and immunoprecipitation techniques suggests that a candidate for the asymmetrically distributed surface antigen in the BBB is the 46K protein. The relationship between the 46K protein and the composition of BBB tight junctions remains to be determined.

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