No abstract

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.CD20 is a B cell-specific tetraspan protein that assembles into oligomeric complexes and forms or regulates a store-operated calcium entry channel that is responsive to B cell receptor (BCR) 1 signaling (1). CD20 is also an effective target for in vivo depletion of malignant or autoimmune B cells using monoclonal antibodies (mAbs), which can activate apoptotic signaling pathways and mediate complement-mediated cytoxicity potentially through mechanisms involving cholesterol-and sphingolipid-rich membrane microdomains known as lipid rafts (2-4). Rafts are thought to function in part as platforms for signaling from those receptors with properties that allow their access to the tightly packed lipid raft environment, which otherwise excludes most membrane proteins (5-7). The operational criteria for assigning raft association of a protein are insolubility in nonionic detergents and buoyancy on density gradients. The detergent best characterized for raft isolation is Triton X-100, and we showed previously that antibodies induce translocation of CD20 from the soluble fraction of Triton X-100 cell lysates into the buoyant insoluble fraction, consistent with its induced association with rafts (8). However, raft association of some proteins can only be demonstrated using very low concentrations of Triton X-100 or other nonionic detergents (9 -11), and we recently found that unligated CD20, although soluble in 1% Triton X-100, was insoluble in 1% Brij 58 (1). Brij 58-insoluble CD20 localized to cholesterol-dependent, buoyant fractions on sucrose density gradients. Importantly, deletion of a short membrane-proximal cytoplasmic sequence, previously shown to be essential for eff...

Nanoparticles have been extensively studied for drug delivery and targeting to specific organs. The functionalization of the nanoparticle surface by site-specific ligands (antibodies, peptides, saccharides) can ensure efficient recognition and binding with relevant biological targets. One of the main challenges in the development of these decorated nanocarriers is the accurate quantification of the amount of ligands on the nanoparticle surface. In this study, nanostructured lipid carriers (NLC) were functionalized with N-acetyl-D-galactosamine (GalNAc) units, known to target the asialoglycoprotein receptor (ASGPR). Different molar percentages of GalNAc-functionalized surfactant (0%, 2%, 5%, and 14%) were used in the formulation. Based on ultra-high-performance liquid chromatography separation and evaporative light-scattering detection (UPLC-ELSD), an analytical method was developed to specifically quantify the amount of GalNAc units present at the NLC surface. This method allowed the accurate quantification of GalNAc surfactant and therefore gave some insights into the structural parameters of these multivalent ligand systems. Our data show that the GalNAc decorated NLC possess large numbers of ligands at their surface and suitable distances between them for efficient multivalent interaction with the ASGPR, and therefore promising liver-targeting efficiency.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or Nacetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimization of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDAapproved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that high density of GalNAc was required to favour hepatocyte internalization via the ASGPR pathway. Interaction studies using surface plasmon resonance and the Macrophage Galactose-Lectin as GalNAc-recognizing lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is a first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.