MPP1-based mechanism of resting state raft organization in the plasma membrane. Is it a general or specialized mechanism in erythroid cells?

Biological membranes are organized in various microdomains, one of the best known being called membrane rafts. The major function of these is thought to organize signaling partners into functional complexes. An important protein found in membrane raft microdomains of erythroid and other blood cells is MPP1 (membrane palmitoylated protein 1)/p55. MPP1 (p55) belongs to the MAGUK (membrane-associated guanylate kinase homolog) family and it is a major target of palmitoylation in the red blood cells (RBCs) membrane. The well-known function of this protein is to participate in formation of the junctional complex of the erythrocyte mem­brane skeleton. However, its function as a “raft organizer” is not well understood. In this review we focus on recent reports concerning MPP1 participation in membrane rafts organization in erythroid cells, including its role in signal transduction. Currently it is not known whether MPP1 could have a similar role in cell types other than erythroid lineage. We present also preliminary data regarding the expression level of MPP1 gene in several non-erythroid cell lines

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins

MPP1 Determines the Mobility of Flotillins and Controls the Confinement of Raft-Associated Molecules

MPP1 (membrane palmitoylated protein 1) belongs to the MAGUK (membrane-associated guanylate kinase homologs) scaffolding protein family. These proteins organize molecules into complexes, thereby maintaining the structural heterogeneity of the plasma membrane (PM). Our previous results indicated that direct, high-affinity interactions between MPP1 and flotillins (raft marker proteins) display dominant PM-modulating capacity in erythroid cells. In this study, with high-resolution structured illuminated imaging, we investigated how these complexes are organized within erythroid cells on the nanometer scale. Furthermore, using other spectroscopic techniques, namely fluorescence recovery after photobleaching (FRAP) and spot-variation fluorescence correlation spectroscopy (svFCS), we revealed that MPP1 acts as a key raft-capturing molecule, regulating temporal immobilization of flotillin-based nanoclusters, and controls local concentration and confinement of sphingomyelin and Thy-1 in raft nanodomains. Our data enabled us to uncover molecular principles governing the key involvement of MPP1-flotillin complexes in the dynamic nanoscale organization of PM of erythroid cells

The Novel Type 1 Fimbriae FimH Receptor Calreticulin Plays a Role in Salmonella Host Specificity

It was suggested that minor differences in the structure of FimH are most likely associated with differences in its adhesion specificities and may determine the tropism of various Salmonella serovars to different species and tissues. We have recently shown that FimH adhesins from host-adapted serovars, e.g., Salmonella Choleraesuis (SCh), bind to other glycoprotein receptors compared to FimH from host-unrestricted Salmonella Enteritidis (SE). Here we identify porcine calreticulin expressed by swine intestinal cells as a host-specific receptor for SCh FimH adhesin, suggesting that such an interaction may contribute to SCh host specificity. Calreticulin was identified by 2D electrophoresis and mass spectrometry as a glycoprotein that was bound specifically by recombinant SCh FimH protein, but not by FimH from SE. The functionality of calreticulin as a specific receptor of SCh FimH adhesin was further confirmed by adhesion and invasion of mutated strains of SCh carrying different variants of FimH proteins to IPEC-J2 cells with overexpression and silenced expression of calreticulin. It was found that SCh carrying the active variant of FimH adhered and invaded IPEC-J2 cells with calreticulin overexpression at significantly higher numbers than those of SCh expressing the non-active variant or SE variant of FimH. Moreover, binding of SCh carrying the active variant of FimH to IPEC-J2 with silenced calreticulin expression was significantly weaker. Furthermore, we observed that SCh infection induces translocation of calreticulin to cell membrane. All of the aforementioned results lead to the general conclusion that Salmonella host specificity requires not only special mechanisms and proteins expressed by the pathogen but also specifically recognized receptors expressed by a specific host