Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.     

Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin

It is generally accepted that ATP is required for intracellular protein breakdown. Reticulocytes contain a soluble ATP-dependent pathway for the degradation of highly abnormal proteins and for the elimination of certain proteins during cell maturation. Reticulocytes and erythrocytes also selectively degrade proteins damaged by oxidation. When these cells were exposed to oxidants, such as phenylhydrazine or nitrite, they showed a large increase in protein breakdown. This oxidant-induced proteolysis was not inhibited in cells depleted of ATP. However, ATP depletion did prevent the degradation of pre-existent cell proteins. In reticulocyte extracts, phenylhydrazine-treated hemoglobin is also degraded rapidly by an ATP-independent process, unlike endogenous proteins and many exogenous polypeptides. This lack of an ATP requirement means that the degradation of oxidant-damaged proteins does not require ligation to ubiquitin (even though phenylhydrazine treatment does make hemoglobin a very good substrate for ubiquitin conjugation). In many respects, the pathway for breakdown of oxidant-treated hemoglobin differs from the ATP-dependent process. The latter has a much higher activation energy than the degradation of oxidized proteins. The ATP-dependent process is inhibited by hemin, 3,4-dichloroisocoumarin, diisopropylfluorophosphate and N-ethylmaleimide. The ATP-independent pathway is less sensitive to N-ethylmaleimide, hemin, and 3,4-dichloroisocoumarin and is not affected by diisopropylfluorophosphate. In addition, only the ATP-dependent proteolytic process is inactivated by dilution or incubation at 37 degrees C in the absence of nucleotides. Reticulocytes thus contain multiple soluble systems for degrading proteins and can rapidly hydrolyze certain types of abnormal proteins by either an ATP-independent or ATP-dependent process. Erythrocytes lack the ATP-dependent process present in reticulocytes; however, erythrocytes retain the capacity to degrade oxidant-damaged hemoglobin. These two processes probably are active in the elimination of different types of abnormal proteins.     

Structural modification of erythrocyte membranes during oxidative stress and activity of membrane bound NADH-methemoglobin reductase

The activity of NADH-methemoglobin reductase (metHb-reductase) in membranes isolated from human erythrocytes treated with phenylhydrazine at its sublytic concentration was studied. A decrease in the activity of membrane-bound metHb-reductase was shown to depend on the concentration of phenylhydrazine. Simultaneously, an increase in the level of membrane-bound methemoglobin and a change in the fluorescence parameters of membrane-bound 4,4'-diisothiocy-anatostilbene-2,2'-disulfonic acid were registered. In the case when Hb-free erythrocyte ghosts were treated with 0.2-2.0 mM phenylhydrazine, the activity of metHb-reductase did not change. The obtained results indicate that the inhibition of the activity of membrane-bound metHb-reductase by phenylhydrazine-induced oxidative stress in human erythrocytes is not caused by the direct action of the oxidant on the enzyme. The reason for this is the interaction of the products of hemoglobin oxidation with erythrocyte membrane (protein band 3) and structural changes in membrane proteins.     

Changes in Brain Protease Activity in Aging

Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age-related changes in calpain specificity and substrate susceptibility were measured. Several age-related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).     

Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils

The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles

Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.     

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns.The rates of breakdown of the substrates were different; expressed as μg of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8–9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate.We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme.These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown.     

Species-specific distribution of cathepsin E in mammalian blood cells

The distribution of cathepsins D and E in leukocytes and erythrocyte ghosts of several mammalian species, and in HL-60 and K-562 cells was examined by means of a combined application of electrophoretic and immunochemical methods. Cathepsin D was found in leukocytes of all species examined, but the distribution of cathepsin E was found to be species-specific: pigs, cows and goats had no cathepsin E activity in leukocytes or erythrocytes at all. In humans, cathepsin E occurred in erythrocytes but not in leukocytes, which contrasted with the guinea pig pattern of its presence in leukocytes and its absence in erythrocytes. No cathepsin E-related enzymes were found in HL-60 or K-562 cells, but these human leukemic cells contained cathepsin D-related enzyme forms that are electrophoretically distinct from normal leukocyte cathepsin D. The present results are inconsistent with the view that cathepsin E may be involved as an essential factor in the biological functions of leukocytes or erythrocytes.     

Altered Membrane Structure and Surface Potential in Homozygous Hemoglobin C Erythrocytes

Background

Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes.

Methodology and Findings

We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was ≈2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO₂ to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes.

Conclusion

Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

Breakdown of brain tubulin by cerebral cathepsin D

The breakdown of cytoplasmic tubulin from brain (purified by ammonium sulfate fractionation and DEAE cellulose chromatography) by cathepsin D from brain (purified by ammonium sulfate fractionation and pepstatin Sepharose chromatography) was studied; changes in the intensity of tubulin gel bands were determined. The pH optimum of hemoglobin breakdown by cathepsin D was 3.2; the pH optimum for tubulin breakdown was 5.8; at pH 5.8 there was no significant hemoglobin breakdown by the enzyme. Tubulin breakdown had an apparent K_m of 1.8 × 10⁻⁵ M and a V_max of 0.56 μg tubulin (μg enzyme per min). The rate of breakdown was heterogeneous and studied on length of incubation; the major portion of tubulin was rapidly broken down and a smaller portion was more stable. The rate under our experimental conditions was 18%/h in the 1–4 h period and 2%/h after 4 h. This was not due to enzyme instability: after 4 h of inhibition freshly added tubulin was rapidly broken down, whereas freshly added enzyme did not increase the rate of breakdown. Thus breakdown heterogeneity was due to substrate (tubulin) heterogeneity. Pepstatin inhibited cathepsin D breakdown of tubulin at acid pH; at pH 7.6 it had no effect. Leupeptin was not inhibitory. We calculated that the cathepsin D content in brain, if fully active, could break down cytoplasmic tubulin with a half-life of 24 h, but it is likely that under in vivo conditions enzyme activity is greatly modified.     

Immunocytochemical determination of membrane-bound IgG using physiologically aged and experimentally aged erythrocytes

The expression of IgG receptor sites at the band 3 protein is important for the recognition and elimination of aged and experimentally altered erythrocytes. Membrane bound IgG was detected in different erythrocyte preparations and microvesicles by means of electron microscopic procedures (protein A-gold-, protein A-gold-silver- and anti-ferritin-sandwich-technique) and light microscopic procedures (immunofluorescence). Physiologically "old", pronase and neuraminidase as well as diamide treated erythrocytes and microvesicles demonstrated significant IgG loading. An increased IgG binding of erythrocytes treated with phenylhydrazine was only evident when higher phenylhydrazine concentrations were used. Both, the alteration of the glycocalyx (conformational changes of the external segment of the glycophorins) and the alteration of the membrane skeleton lead to an unmasking of the IgG receptor site at band 3 proteins (transmembrane effect). The result is an overcritical loading of cells with IgG molecules which initiate the elimination of the erythrocytes by macrophages of the Reticulo-Histiocytic-System.     

Alterations of red cell membranes from phenylhydrazine-treated rabbits

Reticulocytosis was induced in rabbits by two methods: phlebotomy and injection of phenylhydrazine. Normal erythrocytes, reticulocytes from bed rabbits, reticulocytes from phenylhydrazine-treated rabbits, and erythrocytes treated in vitro with phenylhydrazine were compared with respect to their plasma membrane labeling by galactose oxidase and NaB³H₄, and lactoperoxidase-catalyzed incorporation of ¹²⁵I. Normal erythrocyte membranes and membranes from reticulocytes of bled rabbits showed almost identical labeling patterns, the presence of 2–3 glycoproteins with moderate to low mobilities on dodecyl sulfate acrylamide gel electrophoresis. Labeling in the absence of enzyme was negligible. In contrast, the reticulocytes from phenylhydrazine-treated rabbits exhibited a large incorporation of tritium without prior treatment with galactose oxidase. Even after prereduction with unlabeled NaBH₄ to remove this nonspecific labeling, the labeled glycoprotein components found in normal erythrocytes were not detectable. Normal erythrocytes treated in vitro with phenylhydrazine, washed, and labeled with galactose oxidase had labeling patterns, including high nonspecific incorporation of ³H, similar to those observed with in vivo phenylhydrazine treatment.Solubilization of membranes with lithium diiososalicylate followed by partitioning with phenol showed that the same glycoproteins were presented in normal or phenylhydrazine membranes, although only the former extract was labeled by galactose oxidase. Individual carbohydrates from the membranes were analyzed by gas-liquid chromatography and, in the case of hexosamines, on the amino acid analyzer. The results of these analyses indicated a slight decline in galactose content with phenylhydrazine treatment. Reticulocyte membranes from bled rabbits also showed a decrease in galactose content, although it was less pronounced.Most of the label incorporated by nonspecific borohydride labeling of membranes from phenylhydrazine-treated animals was found associated with protein. The modified amino acids from labeled proteins are similar to those formed in reactions of oxidized lipids and proteins in model systems.     

Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family

Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.     

Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells.

The membrane association of the lysosomal enzymes cathepsin D and glucocerebrosidase and its naturally occurring sphingolipid activating protein was studied in HepG2 cells. We differentially permeabilized cells with low concentrations of saponin, at which secretory proteins rinsed out completely, whereas integral membrane proteins were not released. All relevant intracellular compartments were shown to be permeabilized by saponin. Metabolic labeling showed that early precursors of cathepsin D, sphingolipid activating protein, and glucocerebrosidase were completely released from the cells, whereas more than 80% of the high molecular mass intermediates were retained by the cells. Treatment of permeabilized cells with 10 mM mannose 6-phosphate released only 50% of the cell-associated cathepsin D. Glucocerebrosidase remained membrane-associated, but cathepsin D and sphingolipid activating protein were released from the cells after proteolytic processing. Sphingolipid activating proteins and cathepsin D behaved similarly during biosynthesis and showed similar sensitivity to mannose 6-phosphate. The membrane association of the intermediate form of cathepsin D was independent of the presence of N-linked oligosaccharides. Subcellular fractionation on sucrose gradients showed that the lysosomal proteins became membrane-associated probably in the Golgi complex, and that both mannose 6-phosphate-dependent and mannose 6-phosphate-independent membrane association occur in the same compartments. We conclude that, in HepG2 cells, cathepsin D, sphingolipid activating protein, and glucocerebrosidase exhibit MPR-independent membrane association which is acquired in the same compartments beyond the rough endoplasmic reticulum.     

Lysosomal enzyme precursors in human fibroblasts. Activation of cathepsin D precursor in vitro and activity of beta-hexosaminidase A precursor towards ganglioside GM2

Precursors of cathepsin D and beta-hexosaminidase were isolated from secretions of human fibroblasts and their activity was studied with natural substrates. The immunoprecipitated precursor of cathepsin D, Mr 53000, was inactive with radioactive hemoglobin as substrate. At pH 3.8-4.2 an activation of the precursor took place, which was correlated by a reduction in size to Mr 51500. The observed cleavage of cathepsin D precursor in vitro resembles the autocatalytic activation of pepsinogen. The precursor of beta-hexosaminidase A is able to cleave the natural substrate GM2 ganglioside. This reaction, like that of the mature enzyme, depends on the presence of a protein activator, which interacts with the substrate and the enzyme.     

Crystal structure of an activation intermediate of cathepsin E

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.     

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging

The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.     

Cathepsin D is membrane-associated in macrophage endosomes

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.     

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.