Distribution Of Surface Proteins Research Articles

Surface protein distribution in Group B Streptococcus isolates from South Africa and identifying vaccine targets through in silico analysis.

Group B Streptococcus (GBS) is a major cause of pneumonia, sepsis, and meningitis in infants younger than 3 months of age. Furthermore, GBS infection in pregnant women is associated with stillbirths and pre-term delivery. It also causes disease in immunocompromised adults and the elderly, but the highest incidence of the disease occurs in neonates and young infants. At this time, there are no licensed vaccines against GBS. Complete GBS genome sequencing has helped identify genetically conserved and immunogenic proteins, which could serve as vaccine immunogens. In this study, in silico reverse vaccinology method were used to evaluate the prevalence and conservation of GBS proteins in invasive and colonizing isolates from South African infants and women, respectively. Furthermore, this study aimed to predict potential GBS vaccine targets by evaluating metrics such as antigenicity, physico-chemical properties, subcellular localization, secondary and tertiary structures, and epitope prediction and conservation. A total of 648 invasive and 603 colonizing GBS isolate sequences were screened against a panel of 89 candidate GBS proteins. Ten of the 89 proteins were highly genetically conserved in invasive and colonizing GBS isolates, nine of which were computationally inferred proteins (gbs2106, SAN_1577, SAN_0356, SAN_1808, SAN_1685, SAN_0413, SAN_0990, SAN_1040, SAN_0226) and one was the surface Immunogenic Protein (SIP). Additionally, the nine proteins were predicted to be more antigenic than the SIP protein (antigenicity score of > 0.6498), highlighting their potential as GBS vaccine antigen targets.

Effects of Drying Temperature and Relative Humidity on Quality Properties of Chinese Dried Noodles

The influence of the drying conditions on protein structural properties and its impact on Chinese dried noodles (CDN) quality properties is addressed in this study. The CDN were produced under nine different drying conditions utilizing combination of three temperatures (40°C, 60°C, and 80°C) and three relative humidities (65%, 75%, and 85%). The color, texture profile analysis of uncooked and cooked noodles, shrinkage ratio, and cooking quality of CDN were assessed. SEM and FTIR microimaging were investigated to determine the changes in the gluten structural properties. Drying temperature and relative humidity have significant effects on quality characteristics of CDN. However, the influences on different indicators were different. Drying temperature was the main influencing factor of the quality of CDN and protein microstructure. After the drying temperature exceeded 60°C, proteins began to aggregate, and the surface protein distribution became uneven. Compared with cross section, the uniformity of protein distribution on the surface of noodles showed a significant decrease. A high temperature (60°C) could improve the quality of CDN products. The quality of CDN products could be adjusted by the combination of drying temperature and relative humidity.

Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications

Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight – Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5nm thick) at the surface which was readily removed under sputtering with C60. Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2μm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo

Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA i to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA i phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA i lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.

Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane

Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many gram-positive bacteria. The attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved C-terminal LPXTG motif and covalently attaches the protein to the peptidoglycan precursor lipid II. It is unclear how the sorting reaction is regulated spatially and what part sortase localization plays in determining the distribution of surface proteins. This is mainly the result of inadequate immunofluorescence techniques required to resolve these issues in certain bacterial pathogens. Here we describe the utilization of the phage lysin PlyC to permeabilize the cell wall of Streptococcus pyogenes to antibodies, thereby allowing the localization of sortase A using deconvolution immunofluorescence microscopy. We find that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M protein anchoring. Sortase distribution to the new septum begins at a very early stage, culminates during septation, and decays after division is completed. This implies that the sorting reaction is a dynamic, highly regulated process, intimately associated with cell division. The ability to study cytoplasmic and membrane antigens using deconvolution immunofluorescence microscopy will facilitate further study of cellular processes in S. pyogenes.

Actin‐based motility of intracellular bacteria, and polarized surface distribution of the bacterial effector molecules

Several intracellular bacterial pathogens, including species of Listeria, Rickettsia, Shigella, Mycobacteria, and Burkholderia, have evolved mechanisms to exploit the actin polymerization machinery of their hosts to induce actin-based motility, enabling these pathogens to spread between host cells without exposing themselves to the extracellular milieu. Efficient cell-to-cell spread requires directional motility, which the bacteria may achieve by concentrating the effector molecules at one pole of their cell body, thereby restricting polymerization of monomeric actin into actin tails to this pole. The study of the molecular processes involved in the initiation of actin tail formation at the bacterial surface, and subsequent actin-based motility, has provided much insight into the pathogenesis of infections caused by these bacteria and into the cell biology of actin dynamics. Concomitantly, this field of research has provided an opportunity to understand the mechanisms whereby bacteria can achieve a polarized distribution of surface proteins. This review will describe the process of actin-based motility of intracellular bacteria, and the mechanisms by which bacteria can obtain a polarized distribution of their surface proteins.

Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells.

Cholesterol-based membrane microdomains, or lipid rafts, are believed to play important, yet poorly defined, roles in protein trafficking and signal transduction. In polarized epithelial cells, the current view is that rafts are involved in apical but not in basolateral protein transport from the trans-Golgi network (TGN). We report here that cholesterol is required in a post-TGN mechanism of basolateral regionalization. Permanently transfected Madin-Darby canine kidney cells segregated the caveolae/raft-associated high-density lipoprotein scavenger receptor class B type I (SR-BI) predominantly to the basolateral domain where it was constitutively internalized and recycled basolaterally. Acute cholesterol depletion did not significantly alter SR-BI internalization, implying a cholesterol depletion-insensitive endocytic process but instead induced its transcytosis through a protein kinase A (PKA)- and microtubule-dependent mechanism. Forskolin also elicited SR-BI transcytosis. The basolateral distribution of endogenous epidermal growth factor receptor remained unaffected. Strikingly, cholesterol depletion induced PKA activity without increasing the cAMP levels. Thus, our results are consistent with a scenario in which cholesterol-based rafts promote internalization and basolateral recycling of internalized SR-BI whereas a PKA pool sensitive to cholesterol depletion mediates SR-BI transcytosis. Regulated transcytosis of SR-BI may provide an additional mechanism to control cholesterol homeostasis. These results disclose relationships between cholesterol-based rafts and PKA activity operating in a post-TGN mechanism of regulated apical-to-basolateral cell surface protein distribution.

Role of heterotrimeric G proteins in polarized membrane transport.

MDCK cells maintain the polarized distribution of surface proteins mainly by sorting the newly synthesized proteins in the trans-Golgi network (TGN). In order to identify the components of the putative sorting machinery and to study factors that affect the sorting process, we have developed an in vitro system that reconstitutes the transport of viral glycoproteins from the TGN to the apical or basolateral surface. We have used this system to study effects of membrane impermeable reagents (such as peptides and antibodies) on the polarized transport. We observed that reagents affecting the stimulatory class (Gs) of heterotrimeric GTP binding proteins (G proteins) influenced the apical but not the basolateral transport. In contrast, reagents specific for the inhibitory class of G proteins (Gi) affected the basolateral but not the apical transport. These results show that the heterotrimeric G proteins differentially regulate the two pathways of polarized transport. The G proteins may regulate the process of polarized sorting of proteins in a fashion analogous to their role in signal transduction by providing a communication link with the cytosolic side of the membrane.

Chapter 2 Methods to Estimate the Polarized Distribution of Surface Antigens in Cultured Epithelial Cells

This chapter reviews the methods employed to transfect and express foreign genes into epithelial cells. It also determines the polarized surface expression of both endogenous and exogenous plasma membrane proteins. The polarized distribution of surface proteins between the apical and the basolateral surfaces of epithelial cells is the basis of their vectorial function. Retroviral promoters, specifically Moloney murine leukemia virus (MuLV) and Rous sarcoma virus long terminal repeats (LTR) have resulted in high levels of expression of exogenous plasma membrane proteins. The neomycin-resistance marker coupled with selection with G418 has been very useful for the selection of stable transfectants. Polarity can be expressed as a ratio between the amounts of a given molecule in apical and basolateral plasma membrane domains. An important consideration in the analysis of this ratio is the fact that the area of the apical domain is usually a fraction of the area of the basolateral domain. It is important to express polarity as a density ratio—that is, a ratio between the amounts of the molecule per unit surface area in each surface domain. Various methods to estimate the surface polarity of a molecule in expressed epithelial cells include isotopic uptake or binding, electrophysiologic procedures, postsection colloidal gold immunocytochernistry, and EM immunocytochemistry.

Asymmetric distribution of surface proteins in monolayer culture of embryonal carcinoma F9 cells

Embryonal carcinoma F9 cells were labelled with [ 125I]-lactoperoxidase either in monolayer culture or after their dissociation and also as dissociated multilayer aggregates. Two-dimensional gel electrophoresis analysis of iodinated proteins revealed two groups of surface proteins, characteristic of non-attached surface (group A) and of attached surface (group B). The content of group A proteins was 40.7 % in the case of monolayer culture and 10.2 % in the case of multilayer aggregates, as compared to the total value of their common surface proteins. With a direct method for detection of lectin-binding proteins it was shown that three major Concanavalin A-binding proteins belong to group A and one to group B. Two wheat germ agglutinin binding proteins were identified as surface proteins of group B.

Regional differences in distribution of surface proteins over the bull spermatozoa.

Surface-radioiodinated bull spermatozoa were ultrasonicated and fractionated by Percoll-gradient centrifugation. The different fractions obtained were solubilized and analyzed by SDS-polyacrylamide gel electrophoresis. Three fractions containing sperm heads, midpieces, and membranes and small fragments of the principal pieces were obtained. The electrophoresis revealed 5 main peaks representing the radioiodinated surface proteins with molecular weights of 80 000-90 000 (Ia), 68 000-75 000 (Ib), 42 000-47 000 (II), 33 000-37 000 (III) and 15 000-18 000 (V) from the intact spermatozoa as well as from each sperm fragment fraction. The major differences between fractions were in the relative magnitudes of the peaks. The peak II characteristically dominated in the head fraction, but was very small in the midpiece fraction. The results from the present study suggest that the peak II seen in the intact spermatozoa is mainly located on the head plasma membrane and that the differences in the sperm surface properties may be due to the uneven distribution or surface exposure of the proteins.

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. II. Internalization of proteins.

The distribution of surface proteins during phagocytosis by rabbit peritoneal polymorphonuclear leukocytes was studied to determine whether the proteins of the phagocytic vesicles of these differentiated cells were representative of the entire set of plasma membrane proteins. Phagocytosis of bovine serum albumin-diisodecylphthalate emulsion by lactoperoxidase-iodinated rabbit neutrophils was linear over 15-20 min at a rate of 96 microgram oil/min/mg cell protein. This rate was similar to that of unlabeled cells. Incorporation of cell-associated free iodine by endogenous myeloperoxidase during phagocytosis was inhibited by 1 mM cyanide, which had no effect on the rate of particle uptake. The surface of intact neutrophils contained at least 13 iodinated proteins distinguishable by polyacrylamide gel electrophoresis followed by autoradiography. Isolated phagosomes were deficient in six of these proteins. The plasma membrane fraction of these cells was missing five of these same proteins which, however, were enriched in a dense surface fraction (Willinger, M., and F. R. Frankel. J. Cell Biol. 82: 32-44). When experimental conditions were reversed, and the PMNs were labeled after phagocytosis, these five proteins remained on the cell surface, while at least three of the major proteins found on resting cells were depleted. Incubating the cells with colchicine, which has been shown to affect the distribution of some plasma membrane constituents during phagocytosis, had no effect on the distribution of surface proteins in our system. These results indicate that a nonrandom interiorization of lactoperoxidase-labeled surface proteins of polymorphonuclear leukocytes occurs during phagocytosis.

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study.

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.

The influence of lipid state on the planar distribution of membrane proteins in Acholeplasma laidlawii

The distribution of surface proteins within the plane of the cell membrane was investigated using a biotin-avidin-ferritin reagent. This reagent reacts specifically, in the case of Acholeplasma laidlawii , with proteins, and permits their visualization in the electron microscope. A. laidlawii membranes were prepared which had different lipid phase transition-temperature ranges as determined by X-ray diffraction. These membranes were labeled at temperatures above, below and in the middle of their transitions, where the states of the lipids were, respectively, smectic, paracrystalline or a mixture of the two phases. In membranes labeled in either the smectic or paracrystalline phase (above or below the transition), the labeled sites were relatively dispersed, whereas in membranes labeled at temperatures in the mixed phase (mid-transition), patches of high and low density of label were found. In all cases, the distribution of the proteins was dependent upon the temperature relative to the lipid transition rather than the absolute temperature. In addition, the patching phenomenon was found to be freely reversible in experiments in which the temperature was shifted after labeling. To explain these observations, a model is proposed which suggests a phase separation of lipid and the preferential association of protein with either the smectic or paracrystalline state. In order that such a change in protein distribution can occur, protein molecules must be mobile through both the paracrystalline and smectic lipid phases over the time-scale of the experiments. These results suggest that temperature-induced changes in the physical state of membrane lipids can cause changes in the relative location of protein components parallel to the membrane surface.