Microwaves and the cell membrane. III. Protein shedding is oxygen and temperature dependent: evidence for cation bridge involvement

Microwaves (2450 MHz, 60 mW/g) are shown to result in the release or shedding of at least 11 low-molecular-weight proteins (less than or equal to 31,000 Da) from rabbit erythrocytes maintained in physiological buffer. Protein release was detected by gel electrophoresis of cell-free supernatants using sensitive silver staining. This release is oxygen dependent and occurs in 30 min for exposures conducted within the special temperature region of 17-21 degrees C, which is linked to a structural or conformational transition in the cell membrane. Shedding of 26,000 and 24,000 Da proteins is unique to microwave treatment, with enhanced release of 28,000 and less than or equal to 15,000 Da species during microwave compared to sham exposures. Two-dimensional isoelectric focusing further reveals that proteins of less than or equal to 14,000 Da shed during microwave treatment exhibit a pI of 6.8-7.3 not seen in sham-treated cells. Treatment of erythrocytes with a serine-directed protease inhibitor does not prevent release of proteins. However, when erythrocytes are maintained at 17-21 degrees C by conventional heating in the absence of divalent cations, release of 28,000-31,000 and less than or equal to 14,000 Da components is detected. This indicates that cation-bridge stability may be important for release of these proteins. The above results provide evidence that microwaves alter erythrocyte protein composition at temperatures linked to a transition in the cell membrane and that destabilization of salt bridges may play a role in an interaction mechanism for protein release.     

Shedding of the 67-kD laminin receptor by human cancer cells

The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis. © 1996 Wiley-Liss, Inc.     

SheddomeDB: the ectodomain shedding database for membrane-bound shed markers

Background

A number of membrane-anchored proteins are known to be released from cell surface via ectodomain shedding. The cleavage and release of membrane proteins has been shown to modulate various cellular processes and disease pathologies. Numerous studies revealed that cell membrane molecules of diverse functional groups are subjected to proteolytic cleavage, and the released soluble form of proteins may modulate various signaling processes. Therefore, in addition to the secreted protein markers that undergo secretion through the secretory pathway, the shed membrane proteins may comprise an additional resource of noninvasive and accessible biomarkers. In this context, identifying the membrane-bound proteins that will be shed has become important in the discovery of clinically noninvasive biomarkers. Nevertheless, a data repository for biological and clinical researchers to review the shedding information, which is experimentally validated, for membrane-bound protein shed markers is still lacking.

Results

In this study, the database SheddomeDB was developed to integrate publicly available data of the shed membrane proteins. A comprehensive literature survey was performed to collect the membrane proteins that were verified to be cleaved or released in the supernatant by immunological-based validation experiments. From 436 studies on shedding, 401 validated shed membrane proteins were included, among which 199 shed membrane proteins have not been annotated or validated yet by existing cleavage databases. SheddomeDB attempted to provide a comprehensive shedding report, including the regulation of shedding machinery and the related function or diseases involved in the shedding events. In addition, our published tool ShedP was embedded into SheddomeDB to support researchers for predicting the shedding event on unknown or unrecorded membrane proteins.

Conclusions

To the best of our knowledge, SheddomeDB is the first database for the identification of experimentally validated shed membrane proteins and currently may provide the most number of membrane proteins for reviewing the shedding information. The database included membrane-bound shed markers associated with numerous cellular processes and diseases, and some of these markers are potential novel markers because they are not annotated or validated yet in other databases. SheddomeDB may provide a useful resource for discovering membrane-bound shed markers. The interactive web of SheddomeDB is publicly available at http://bal.ym.edu.tw/SheddomeDB/.

     

Isolation and partial characterization of the human erythrocyte band 7 integral membrane protein.

Monoclonal antibodies to the Mr 31,000 major integral membrane protein of the human erythrocyte band 7 region were used to identify the corresponding polypeptide chain and epitope-carrying fragments on immunoblots. Analysis of the erythrocyte membrane, membrane fractions, and cytosol revealed that the Mr 31,000 band 7 integral membrane protein is unique and not related to any of the other water-soluble or membrane-bound band 7 components. Cross-reacting proteins were identified in the membranes of other mammalian erythrocytes and in cell lines of epithelial and lymphoid origin. Proteolytic digestion of intact human erythrocytes or erythrocyte membranes demonstrated that the band 7 integral membrane protein has an intracellular domain larger than Mr 12,000; it does not have an extracellular one. One of the monoclonal antibodies was employed for the isolation of band 7 integral membrane protein by immunoaffinity chromatography; subsequent Edman degradation revealed a blocked N-terminus.     

Microwave bioeffects in the erythrocyte are temperature and pO2 dependent: Cation permeability and protein shedding occur at the membrane phase transition

Microwave exposure (2450 MHz, 60 mW/g, CW) of rabbit erythrocytes increases Na passive transport only at membrane phase transition temperatures (T_c) of 17–19°C. This permeability effect is enhanced for relative hypoxia which is characteristic of intracellular oxygen tension (pO₂ ? 5 mm Hg). Neither the permeability nor the pO₂ effects are observed in temperature-matched (± 0.05°C), sham-exposed controls. In addition, at T_c, microwave exposure is observed to induce the shedding or release of two erythrocyte proteins not seen in sham-exposed controls. Moreover, the enhanced shedding of at least seven other proteins all of molecular weight ? 28,000 D was detected in the microwave-treated samples. Using sensitive silver staining we estimate that approximately 450 fg of protein were shed per erythrocyte. These results demonstrate that temperature and pO₂ are important influences on both functional and structural responses of cell membranes to microwave radiation.     

Surface NADH oxidase of HeLa cells lacks intrinsic membrane binding motifs

Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37 degrees C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.     

Marginal product pricing in the ecosystem.

Surface membrane biosynthesis and turnover is reviewed focusing mainly on the fate of cell surface constituents after they terminated their sojourn as part of a functional cell structure. The different experimental approaches to study this problem are described and original data are presented on the turnover of surface membrane constituents of chicken embryo cells in culture. It is proposed that as a consequence of surface membrane turnover, certain surface macromolecules are continuously shed from cells. The size and charge of these molecules was found to be identical to molecules released from cells by mild trypsin treatment. The term shedding is proposed for this process which is assumed to occur both in vitro and in vivo. Many systems in which shedding of cell surface constituents is clearly demonstrated or can be tentatively suggested are described. The biological significance of cell surface carbohydrate containing macromolecules and the possible role of these shed cellular entities is discussed.     

Human erythrocyte transglutaminase: purification and preliminary characterisation

Erythrocyte transglutaminase was purified by anion-exchange chromatography, size exclusion and affinity chromatography. Homogeneity was achieved by an additional step of HPLC size-exclusion chromatography. The molecular mass of the purified enzyme was calculated to be 65,000 Da by size-exclusion chromatography and sucrose-gradient centrifugation, and 92,000 Da by SDS-PAGE, thus suggesting a high degree of asymmetry. The amino-acid composition of erythrocyte transglutaminase differed substantially from that of the guinea-pig liver enzyme, notably with respect to the number of histidine, cysteine and acidic amino-acid residues. The enzyme has an absolute requirement for divalent cations for activity: calcium, manganese, and the lanthanides terbium and gadolinium activate the enzyme in decreasing order of efficacy, while no activity is displayed in the presence of magnesium. In the presence but not in the absence of calcium ions, the enzyme is rapidly inactivated by N-ethylmaleimide and by diethylpyrocarbonate suggesting that the cation influences the reactivity of amino acids essential for catalysis. When erythrocyte proteins are employed as amine acceptors in the presence of calcium, the erythrocyte transglutaminase appears to preferentially modify membrane-associated proteins, although, in the absence of calcium ions and exogenous amines, it displays a pH-dependent interaction with soluble proteins.     

A proteomic approach to characterize protein shedding

Shedding (i.e. proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fractions, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 45 membrane-associated proteins were identified including 22 that contain at least one transmembrane domain and 23 that indirectly associate with the extracellular surface of the plasma membrane. Of the 22 transmembrane proteins, 18 were identified by extracellular peptides providing strong evidence they originate from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified two proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78 kDa glucose-regulated protein and lactate dehydrogenase A. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78 kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that tandem mass spectrometry can be used to identify shed proteins and to estimate changes in protein abundance.     

Microwaves and the cell membrane. II. Temperature, plasma, and oxygen mediate microwave-induced membrane permeability in the erythrocyte

Microwaves (2450 MHz) are shown to increase 22Na permeability of rabbit erythrocytes for exposures only within the narrow temperature range of 17.7 to 19.5 degrees C (Tc) which coincides with a nonlinearity in the Arrhenius plot reflecting an apparent membrane phase transition. Significantly, this response is not observed for cholesterol-loaded erythrocyte membranes which exhibit a linear Arrhenius plot and no apparent phase transition at Tc. The permeability increase at Tc is a nonlinear function of absorbed power but is a linear function of the internal electric field strength of the sample and saturates at approximately 400 mW/g and 600 V/m, respectively. The permeability increase was found to be reversible and transient in that immediately following termination of exposure sodium influx is significantly reduced but returns to normal within 60 min. Extracellular factors exert a significant influence on the microwave effect. The presence of plasma markedly potentiates the increase in 22Na permeability at Tc. Oxygen also modulates the microwave effect with relative hypoxia (5 mm Hg) and hyperoxia (760 mm Hg) enhancing the permeability increase. In contrast, the presence of two antioxidants, ascorbic acid or mercaptoethanol, inhibits the effect. These findings raise important questions about the physical and chemical nature of microwave interactions with cell membranes and also shed light on earlier studies reporting either positive or negative effects on membrane permeability.     

Metalloprotease-mediated shedding of enzymatically active mouse ecto-ADP-ribosyltransferase ART2.2 upon T cell activation

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-alpha-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.     

GPI-linked proteins do not transfer spontaneously from erythrocytes to liposomes. New aspects of reorganization of the cell membrane

Exposure of cells to liposomes results in the release of integral membrane proteins. However, it is still controversial whether the release is due to spontaneous protein transfer from cells to liposomes or shed vesicles released from cells. We investigated this issue in an erythrocyte-liposome system by examining the location of acetylcholinesterase (AChE, an integral membrane protein marker), cholesterol (erythrocyte membrane lipid marker), hemoglobin (cytosolic protein marker), and a nonexchangeable lipid marker in liposomes in a sucrose density gradient at high resolution. The density distribution showed that AChE is not transferred to the liposomes but is located on small (about 50 nm) light (10-20 wt % sucrose) or large (about 200 nm) heavy shed vesicles (more than 30 wt % sucrose). AChE in the light shed-vesicle fraction markedly increased even after its level in the heavy fraction reached a plateau. AChE was also released from isolated heavy shed vesicles and accumulated in the small light shed-vesicle fraction in the presence of liposomes. After incubation of spherical erythrocytes (morphological index, 5.0) with liposomes, AChE hardly appeared in the heavy shed-vesicle fraction, and the majority (>99%) appeared in the light shed-vesicle fraction, indicating that AChE is released from both the erythrocytes and heavy shed vesicles to the light shed-vesicle fraction, which becomes rich in AChE. Our results demonstrated for the first time that GPI-linked proteins do not spontaneously transfer from erythrocytes to liposomes. Our study also suggests that in vivo GPI-linked membrane proteins do not spontaneously transfer between cell membranes but that some catalyst is needed.     

Undifferentiated and differentiated L6 myoblast plasma membranes. I: Comparison of the morphology of plasma membrane vesiculation and the factors influencing the vesiculation process

Monolayer cultures of a variety of cell lines can be induced to shed plasma membrane vesicles by exposure to 25 mM formaldehyde--2 mM dithiothreitol. An analysis of the process of membrane shedding can provide a method to probe the general characteristics of the plasma membrane in different cell populations. We have, therefore, analyzed membrane shedding in L6 myoblasts at different stages of differentiation. The results show that the morphology of membrane shedding is comparable in undifferentiated or differentiated L6 myoblasts, as are the factors that influence the process of membrane shedding. L6 myoblasts at various stages of differentiation have similar requirements for metabolic energy, physiological temperature, pH, and monovalent and divalent cations.     

Mechanism of selective release of membrane proteins from human erythrocytes in the presence of liposomes

Incubation of erythrocytes with liposomes results in the release of shed vesicles rich in glycosyl-phosphatidylinositol (GPI)-anchored proteins but poor in transmembranous proteins. We investigated the mechanisms of membrane protein polarization by examining the effect of the interaction between spectrin and membrane proteins on the release of a transmembranous protein, band 3, and a GPI-anchored protein, acetylcholinesterase (AChE), from erythrocyte ghosts. Polymerization of spectrin resulted in a 30-fold decrease in the released amount of band 3 per constant amount of shed vesicles but did not affect the amount of released AChE per constant amount of shed vesicles. On the other hand, the amount of released band 3 per constant amount of shed vesicles increased by cleaving the cytoplasmic part of band 3. Our results first demonstrated that the diffusibility of membrane proteins determined by steric hindrance between membrane proteins and protein mesh primarily determines the ease of localization of membrane proteins into shed vesicles. Taken together with the recent biophysical studies, we built a "fence selection model" that retrograding spectrin mesh sweeps diffusing band 3 molecules from the tip of the membrane crenated area toward the entry of the crenated area, but not AChE molecules. Our study describes a novel method for isolation of a large number of vesicles containing special and intact membrane proteins from cells not by using detergents or organic solvents, but by utilizing the fence effect between the cytoskeleton and membrane proteins.     

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.     

The cell surface proteoglycan of mouse mammary epithelial cells. The extracellular domain contains N terminus and a peptide sequence present in a conditioned medium proteoglycan

The cell surface proteoglycan of mouse mammary epithelial (NMuMG) cells behaves as a receptor for interstitial matrix materials and consists of a membrane-associated domain and an extracellular domain (ectodomain). The ectodomain can be released intact from the cell surface by mild trypsin treatment and appears to be shed from the cells into the culture medium by cleavage from the membrane-associated domain. We have examined the chemical relationship between the trypsin-released proteoglycan and shed proteoglycan to assess their relationship to each other and to the cell surface. Purification and amino acid sequencing of the ectodomain released by mild trypsin treatment resulted in no clear signal until the protein was cleaved by CNBr treatment, suggesting that its N terminus is blocked and oriented extracellularly. The amino acid sequence identified in the trypsin-released ectodomain is present near the N terminus of the shed proteoglycan purified from conditioned medium, indicating that both forms possess closely related (if not identical) core proteins. The sequence reveals a pentapeptide identical to one near the C terminus of the rat hepatic lectin (RHL-1, rat asialoglycoprotein receptor). The medium proteoglycan, which migrates as a smear on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (between 93 and 200 kDa), is heterogeneous due to varying amounts of glycosaminoglycan and substituted O-linked oligosaccharide present on an approximately 46-kDa polypeptide.     

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.     

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.     

A proteomic approach for the identification of cell-surface proteins shed by metalloproteases

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.