Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments

Abstract

Lipid biosensors are robust tools used in both in vitro and in vivo applications of lipid imaging and lipid detection. Lactadherin C2 (LactC2) was described in 2000 as being a potent and specific sensor for phosphatidylserine (PS) (Andersen et al. Biochemistry 39:6200-6206, 2000). PS is an anionic phospholipid enriched in the inner leaflet of the plasma membrane and has paramount roles in apoptosis, cells signaling, and autophagy. The myriad roles PS plays in membrane dynamics make monitoring PS levels and function an important endeavor. LactC2 has functioned as a tantamount PS biosensor namely in the field of cellular imaging. While PS specificity and high affinity of LactC2 for PS containing membranes has been well established, much less is known regarding LactC2 selectivity for subcellular pools of PS or PS within different membrane environments (e.g., in the presence of cholesterol). Thus, there has been a lack of studies that have compared LactC2 PS sensitivity based upon the acyl chain length and saturation or the presence of other host lipids such as cholesterol. Here, we use surface plasmon resonance as a label-free method to quantitatively assess the apparent binding affinity of LactC2 for membranes containing PS with different acyl chains, different fluidity, as well as representative lipid vesicle mimetics of cellular membranes. Results demonstrate that LactC2 is an unbiased sensor for PS, and can sensitively interact with membranes containing PS with different acyl chain saturation and interact with PS species in a cholesterol-independent manner.

Proteins and Ions Compete for Membrane Interaction: the case of Lactadherin

“…To our knowledge, this is the first report on the binding affinity of lactadherin using nanodiscs as the membrane model. We find that the binding affinity in the absence of Ca 2+ is 296.2 ± 82.8 nM, which is in agreement with previously reported literature (43). In order to more accurately model a physiologically relevant system, we also measured the binding affinity of lactadherin in the presence of Ca 2+ ; which was measured to be 283.0 ± 66.5 (nM).…”

“…As reported previously (43), the binding isotherms were plotted from maximal steady-state response units versus the protein concentration flowed over the chip surface, from which the equilibrium binding constant, Kd, values were derived by fitting the single-site ligand binding equation to the data in Graph Pad Prism 8.0 (44). The single-site ligand binding equation is:…”

Flagging fusion: Phosphatidylserine signaling in cell–cell fusion

“… Adenosine and β-adrenergic receptors Nonselective, constitutive, bidirectional scramblases ( 193 ) ? P4-ATPases of the ATP 8, 9, 10, and 11 families Flip PS from the exofacial to the cytofacial leaflet of the PM ( 61 ) Myoblasts ( 169 ) ABC transporter CED-7 Elicits PS exposure on axon PMs in C. elegans ( 194 ) Axon regeneration ( 127 ) PLSCRs May play some role in eliciting PS exposure ( 79 , 81 ) Viral entry ( 82 ) PS recognizing machinery Annexins Soluble, PS-binding proteins that function as assembly factors in many biological processes ( 195 ) Myoblasts ( 21 , 196 ), trophoblasts ( 167 ), osteoclasts ( 22 ) Lactadherin Soluble, PS-binding protein ( 197 ) Sperm-egg ( 168 ) Protein S Soluble, PS-binding protein ( 198 ) ? GAS-6 Soluble, PS-binding protein ( 177 ) Viral entry ( 199 ) CD300 receptors Membrane-bound receptors with affinity for exofacial lipids, some specifically bind PS ( 200 ) Viral entry ( 201 ) TIM receptors 1 and 4 Membrane-bound PS receptors with major roles in immunity ( 202 ) Viral entry ( 203 ) BAI receptors 1 and 3 Membrane-bound PS receptors ( 204 ) Myoblasts ( …”

Regulation of membrane homeostasis by TMC1 mechanoelectrical transduction channels is essential for hearing

“…It is important to note that due to the Ca 2+ -dependent PS binding of AnV, cochlear explants were incubated for 5 min in HBSS containing AnV after BAPTA-treatment. Although the regeneration of the tip links takes hours (Zhao et al, 1996), we wanted to confirm these results using a recombinant C2 domain of lactadherin/MFG-E8 fused to the fluorescent protein clover (clover-Lact-C2), which binds PS in the absence of Ca 2+ (Del Vecchio and Stahelin, 2018). Consistent with the AnV results, clover-Lact-C2 labeling was detected in BAPTA-treated cells but not in the control (Figure S3), further supporting the specific detection of externalized PS in response to tip link disruption.…”