Cleveland, E. | Search bioRxiv

bioRxiv2026

Design of Fluorescent Membrane Scaffold Proteins for Nanodiscs

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications in structural, functional, and biophysical studies. The original MSP was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including fluorescent nanodiscs of varying designs. However, chemical derivatization with fluorophores can be expensive, and prior designs of split fluorescent proteins fused to MSP were limited in the color pallet available. Here, we developed MSPs with a wide range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. They are also suitable for assembly of nanodiscs with embedded integral membrane protein. Finally, we apply these constructs to monitor peripheral membrane protein binding to lipids via fluorescence resonance energy transfer. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v3_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1bf2d2borg.highwire.dtl.DTLVardef@2ea07eorg.highwire.dtl.DTLVardef@12aeccforg.highwire.dtl.DTLVardef@121430d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Shear Stress Promotes Remodeling of Platelet Glycosylation via Upregulation of Platelet Glycosidase Activity: The Ulterior Cause of MCS-Related Thrombocytopenia?

ObjectiveMechanical circulatory support (MCS) is a mainstay of therapy for advanced and end-stage heart failure. Accompanied by systemic anticoagulation, contemporary MCS has become less thrombogenic, with bleeding complications emerging as a major cause of readmission and 1-year mortality of device-supported patients. Shear-mediated platelet dysfunction (SMPD) and thrombocytopenia of undefined etiology are primary drivers of MCS-related bleeding. Recently, it has been demonstrated that deprivation of platelet surface glycosylation is associated with the decline of hemostatic function, microvesiculation, and premature apoptosis. We tested the hypothesis that shear stress induces remodeling of platelet surface glycosylation via upregulation of glycosidase activity, thus facilitating platelet count decline and intense microvesiculation. Approach and ResultsHuman gel-filtered platelets were exposed to continuous shear stress in vitro. Platelets and platelet-derived microparticles were quantified via flow cytometry using size standard fluorescent nanobeads. Platelet surface glycosylation was evaluated using lectin staining and multicolor flow cytometry; lectin blotting was utilized to verify glycosylation of individual glycoproteins. Platelet neuraminidase, galactosidase, hexosaminidase, and mannosidase activities were quantified using 4-methylumbelliferone-based fluorogenic substrates. We demonstrated that shear stress promotes selective remodeling of platelet glycosylation via downregulation of 2,6-sialylation, terminal galactose, and mannose, while 2,3-sialylation remained largely unchanged. Shear-mediated deglycosylation is partially attenuated by neuraminidase inhibitors DANA and zanamivir, strongly suggesting involvement of platelet neuraminidase in observed phenomena. Platelets exhibited high basal hexosaminidase and mannosidase activities; basal activities of platelet neuraminidase and galactosidase were rather low and were significantly upregulated by shear stress. Shear stress of increased magnitude and duration potentiated an incremental decline of platelet count and immense microvesiculation, both being further exacerbated by neuraminidase. ConclusionsOur data indicate that shear stress accumulation, consistent with supraphysiologic conditions of device-supported circulation, promotes remodeling of platelet glycosylation via selective upregulation of platelet glycosidase activity. Shear-mediated platelet deglycosylation is associated with platelet count drop and increased microvesiculation, thus offering a direct link between deglycosylation and thrombocytopenia observed in device-supported patients. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/583630v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@84eda2org.highwire.dtl.DTLVardef@10d3f90org.highwire.dtl.DTLVardef@c83bcborg.highwire.dtl.DTLVardef@1cd981c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗