Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Tarone, Guido; Hamasaki, N; Fukuda, Minoru; Marchesi, V.

doi:10.1007/bf01869253

Cited by 52 publications

(8 citation statements)

References 20 publications

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“…The presence of proteolytic activity associated with the red blood cell has been known for some time (25, 26). Before the discovery of the proteasome and substantial evidence supporting its role as the major proteolytic machinery of the eukaryotic cell, a number of reports described the existence of an ATP-independent proteolytic activity in the cytosol of the human erythrocyte (27–30).…”

Section: Discussionmentioning

confidence: 99%

Antimicrobial peptides are degraded by the cytosolic proteases of human erythrocytes

Starr¹,

Wimley²

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Well-studied and promising antimicrobial peptides (AMPs), with potent bactericidal activity, in vitro, have yet to have a significant impact in human medicine beyond topical applications. We previously showed that interactions of AMPs with concentrated human erythrocytes inhibit many of them, and suggested that screens and assays should be done in their presence to mimic host cell inhibition. Here, we use AMPs to characterize the activity of proteases that are associated with human erythrocytes. The representative AMPs, ARVA and indolicidin, are degraded significantly during incubation with dilute, washed erythrocytes and yield a variety of degradation products, suggesting significant exopeptidase activity. Comparison of these fragments with those obtained from incubation with serum shows that the proteolytic activity associated with cells yields unique products that are not explained by residual serum proteases. By separately testing the membrane and cytosolic fractions, we show that erythrocyte proteolytic activity is found only in the cytosol. Finally, we incubated a diverse cross-section of natural and synthetic linear AMPs with human erythrocyte cytosolic extracts and observed degradation of all of them. These results show that, in addition to cell binding, proteolysis can also contribute significantly to host cell inhibition of AMPs in vitro and possibly also in vivo.

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Section: Discussionmentioning

confidence: 99%

Antimicrobial peptides are degraded by the cytosolic proteases of human erythrocytes

Starr¹,

Wimley²

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Protein degradation in human erythrocyte membrane has also been reported, mostly for ghosts incubated for many hours (21)(22)(23)(24)(25). Various Ca2e-dependent and Ca2e-independent proteases have been identified and isolated from human erythrocytes (26)(27)(28)(29).…”

Section: Discussionmentioning

confidence: 99%

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Kosower¹,

Glaser²,

Kosower³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membrane components from certain constraints. In addition, the motion of membrane components precedes fusion and must be promoted by reagents such as A2C, leading to the creation of fusion-potent lipid areas. This sequence of chemical and physical events occurs in other fusion processes.Membrane fusion is a ubiquitous cellular process, mediating such phenomena as fertilization, exocytosis, endocytosis, phagocytosis, and turnover of intracellular organelles (1, 2). Fusion can be induced experimentally by certain viruses (3) and chemical agents (4, 5). Membrane-mobility agents are long-chain esters designed to promote motion of molecules in cell membranes. Membrane-mobility agents, which enter cells via the small particles formed on dispersion in aqueous medium, promote the lateral mobility of certain ligand-membrane receptor complexes and inhibit cytokinesis in some types of cells (5-9). Membrane-mobility agents, especially 2-(2-methoxyethoxy)ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C), are efficient promoters of cell fusion; the results obtained using a defined agent such as A2C have allowed the identification of stages in the overall fusion process and the formulation of a molecular mechanism for membrane fusion (10-13).We have previously found differences in the occurrence of A2C-induced fusion among various mammalian non-nucleated erythrocytes as well as among avian nucleated erythrocytes (12, 13): erythrocytes of some species fuse easily, whereas those of others do not. "Mixed fusion," that between fusible erythrocytes from different species, is promoted by A2C. Moreover, the potential for fusion is a transferable characteristic, fusion being induced by A2C in otherwise nonfusible cells if mixed with fusible erythrocytes (13). The molecular basis for the differences observed among the erythrocytes of various species and for the transfer of fusibility has not yet been clarified.We now show that the difference between fusible rat erythrocytes and the nonfusible human cells is correlated with proteolytic activity; degradation of rat cell membrane proteins is caused by a Ca2'-activated cytoplasmic protease(s). The human erythrocyte is deficient in this protease activity. Membrane protein degradation is shown to be a necessary, but not sufficient, prerequisite for membrane fusion. Release of membrane components from constraints imposed by the cytoskeleton, allowing membrane component mobility, is proposed as one of the steps in the fusion process. Furthermore, the mobility of the membrane components must be promoted by...

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“…IL, respectively, by proteolysis during the preparation of the sialoglycoproteins . Several neutral (3,15,26,27,41,42) and acid proteases (14,17,19,28,31) have been detected in preparations of erythrocyte ghosts, arising probably from contaminating leukocytes (15). Some of the neutral proteases have essential sulfhydryl groups (27) and perhaps are partially inactivated by STT like other enzymes with essential sulfhydryls (21,29,30).…”

Section: Discussionmentioning

confidence: 99%

Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts.

Sarris¹,

Palade²

1982

The Journal of Cell Biology

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With the lithium diiodosalicylate (LIS) extraction-phenol partition method, we have isolated a sialoglycoprotein fraction from DBA/2 mouse erythrocyte ghosts . We have demonstrated that the Laemmeli system for SDS PAGE can resolve this fraction into four monomers of which two (gp-2.1 and gp-3 .1) appear to be authentic, whereas the other two (gp-2 .2 and gp-3 .2) are probably generated from gp-2 .1 and gp-3 .1, by limited proteolysis during the isolation procedure. All four components contain O-acetylated neuraminic acid residues, can be stained with Periodic acid-Schiff reagent (PAS) and with Coomassie Brilliant Blue (CB), and can be radioiodinated with the lactoperoxidase-glucose oxidase (LPO-GO) method . All monomers but especially gp-2 .1 and gp-3 .1 generate characteristic aggregates during solubilization in SDS. The aggregation is enhanced by boiling at high concentrations, and can be reversed by boiling at low concentrations . In addition, the fraction contains a diffuse component present also in ghosts which stains poorly with CB and with PAS and cannot be radioiodinated by the LPO-GO technique. SDS PAGE in the Steck and Yu gel system does not give an accurate separation of the sialoglycoprotein monomers .In our previous work on murine erythrocyte ghosts, we detected three sialoglycoproteins by Periodic acid-Schiff reagent (PAS) staining after SDS PAGE carried out according to Steck and Yu (37), and we presented evidence for the existence and preferential localization of O-acetylated sialyl residues on two of these three sialoglycoproteins (32) . When the sialoglycoproteins were subsequently analyzed on Laemmli gels (20), however, we obtained a very different pattern. The discrepancy raised the following questions : Which of the visible bands are sialoglycoprotein monomers and which are aggregates? Which of the gel systems truly separates the monomers? And, if polymerization occurs, what are the conditions that can prevent it in vitro?Here we partially answer these questions. We eluted all resolved components from each of the two gel systems and reran each component in the same or the alternative system . Each band will be named by a prefix gp, (for glycoprotein), and a suffix: S to signify initial separation on a Steck and Yu gel ; or L to signify separation on a Laemmli gel. The prefixes will always be used in the text but may be omitted from the figures . Gel Electrophoresis SDS PAGE was performed by solubilizing the proteins in 62 .5 mM Tris-HCI, pH 6.8, 3% wt/vol SDS, 2 M urea, 2 mM EDTA, and either 20 mM dithiothreitol (DTT) or 1 .5% (vol/vol) ß-mercaptoethanol, heating the mixture in a boiling water bath for 5 min, and then separating its components according to Laemmli (20) . Some analyses were also performed on Steck and Yu gels (37) as in Sarris and Palade (32) by solubilizing the preparations in 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 3% (wt/vol) SDS, 1.5% (vol/vol) ß-mercaptoethanol and 6% (vol/ vol) glycerol, and heating them in a boiling water bath for 5 min before electrophoretic se...

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Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Cited by 52 publications

References 20 publications

Antimicrobial peptides are degraded by the cytosolic proteases of human erythrocytes

Antimicrobial peptides are degraded by the cytosolic proteases of human erythrocytes

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts.

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