Andrew G. Booth

Rabbit peroxidase-antiperoxidase complex (PAP) as a model for the uptake of immunoglobulin G by the human placenta

SummaryRabbit peroxidase-antiperoxidase complex (PAP) has been shown to bind to IgG receptors on the human placental syncytiotrophoblast microvillar membrane. Its binding characteristics suggest that it is suitable as a probe for studies on the uptake of IgG by the human placenta.A novel assay system was developed to measure the dissociation constants (Kd) of the binding of PAP and of unlabelled human IgG to purified placental microvillar membranes. TheKd for PAP was found to be 54 nM, while that for unlabelled IgG was found to be 17.5 nM.The uptake of PAP by placental tissue slices was observed using peroxidase histochemistry and electron microscopy. In initial experiments, reaction product was confined to the peripheral regions of the syncytiotrophoblast. Assaying a placental homogenate for catalase activity showed that it contained 250 units of activity per g wet weight of tissue (compared with 680 units/g for rat liver). Treatment of fixed tissue with the catalase inhibitor 3-amino-1, 2, 4-triazole allowed the localization of peroxidase reaction product in deeper regions of the syncytiotrophoblast. Based on observations of the localization of reaction product, we propose that PAP is taken up in coated pits, transferred into large apical multivesicular bodies, segregated into small vesicles which then transport it to the Golgi. From here the PAP is directed to the basal membrane by a mechanism as yet unknown.

Immunoelectrophoretic evidence that the human syncytiotrophoblast transferrin receptor is identical to a major plasma membrane antigen present throughout pregnancy

Crossed immunoelectrophoresis of solubilized syncytiotrophoblast microvillar proteins has been performed through intermediate gels, containing a monospecific antiserum to transferrin, into gels containing an antiserum raised to purified microvilli from term placentae. This technique resolved the microvillar transferrin-receptor complex into its component proteins. Crossed hydrophobic interaction immunoelectrophoresis confirmed that the transferrin receptor is an amphiphilic integral membrane protein. It is also a major antigen of the microvillar membrane and is present throughout pregnancy.

PROTEINS OF THE KIDNEY MICROVILLAR MEMBRANE: TOPOLOGICAL CONSIDERATIONS

Abstract 1. 1. A method for the asymmetric labelling of pig kidney microvillar membrane proteins is described. 2. 2. The photo-activated reagent, 3,5-di[125I]iodo-4-azido-benzene sulphonate, enabled four of the membrane peptidases to be characterized as transmembrane proteins.

Membrane remodelling by a lipidated endosomal sorting complex required for transport-III chimera, in vitro

The complexity of eukaryotic cells is underscored by the compartmentalization of chemical signals by phospholipid membranes. A grand challenge of synthetic biology is building life from the ‘bottom-up’, for the purpose of generating systems simple enough to precisely interrogate biological pathways or for adapting biology to perform entirely novel functions. Achieving compartmentalization of chemistries in an addressable manner is a task exquisitely refined by nature and embodied in a unique membrane remodelling machinery that pushes membranes away from the cytosol, the ESCRT-III (endosomal sorting complex required for transport-III) complex. Here, we show efforts to engineer a single ESCRT-III protein merging functional features from its different components. The activity of such a designed ESCRT-III is shown by its ability to drive the formation of compartments encapsulating fluorescent cargo. It appears that the modular nature of ESCRT-III allows its functional repurposing into a minimal machinery that performs sophisticated membrane remodelling, therefore enabling its use to create eukaryotic-like multi-compartment architectures.

In vitro membrane remodelling by ESCRT-II/ESCRT-III is regulated by negative feedback from membrane tension

Artificial cells can shed new light on the molecular basis for life and hold potential for new chemical technologies. Inspired by how nature dynamically regulates its membrane compartments, we aim to repurpose the endosomal sorting complex required for transport (ESCRT) to generate complex membrane architectures as suitable scaffolds for artificial cells. Purified ESCRT-III components perform topological transformations on giant unilamellar vesicles (GUVs) to create complex “vesicles-within-a-vesicle” architectures resembling the compartmentalisation in eukaryotic cells. Thus far, the proposed mechanisms for this activity are based on how assembly and disassembly of ESCRT-III on the membrane drives deformation. Here we demonstrate the existence of a negative feedback mechanism from membrane mechanics that regulates ESCRT-III activity. ILV formation removes excess membrane area, increasing tension, which in turn suppresses downstream ILV formation. This mechanism for in vitro regulation of ESCRT-III activity may also have important implications for its in vivo functions.