Isolation and characterization of the lytic enzyme from Bacillus subtilis 797

The isolation procedure and some properties of the lytic enzyme produced by Bacillus subtilis 797 and capable of hydrolyzing the E. coli K-12 cells are described. Using hydrophobic chromatography on DNP-hexamethylene diamine Sepharose 4B and ion-exchange chromatography on DEAE-cellulose, a highly purified enzyme preparation with mol. weight of 28000, pI 8.2 has been obtained. The amino acid composition of the enzyme has been determined: Asp--37, Thr--17, Ser--34, Glu--15, Pro--14, Gly--17, Ala--36, Val--28, Met--4, Ile--11, Leu--17, Tyr--9, Phe--4, Lys--18, His--5, Arg--4. The enzyme is inhibited by a specific inhibitor of serine proteinases--benzylsulfonylfluoride, and is insensitive to EDTA and iodoacetic acid. The lytic enzyme has a proteolytic activity and splits the peptide substrate of bacterial serine proteinases--p-nitroanilide benzyloxycarbonyl-L-analyl-L-alanyl-L-leucine; the maxima for both activities lie within the pH range of 7.8-8.5. The lytic protease has the highest stability at pH 6-10. In some of its properties the enzyme is similar to serine proteinase from Bac. subtilis, i. e. subtilisins.     

The study of Escherichia coli proteases. Intracellular serine protease of E. coli-an analogue of bacillus proteases.

Two serine proteases in extracts of Escherichia coli grown to stationary phase were purified to homogeneity using affinity chromatography on gramicidin S-Sepharose 4B. One enzyme was closely related to, if not identical with, the 'trypsin-like' protease II of E. coli. The other was capable of cleaving the subtilisin chromogenic substrate N-carbobenzoxy-L-alanyl-L-alanyl-L-leucine-p-nitroanilide and resembled the intracellular serine proteases of Bacillus spp. The amino acid composition of this E. coli protease was similar to that of the Bacillus licheniformis enzyme. These data indicate a relationship between proteolytic enzymes of evolutionary distant Gram-negative Enterobacteriaceae and Gram-positive spore-forming Bacillus.     

Subtilisin like protease secreted in Bacillus pumilus KMM 62 on different growth stages

A protease secreted in Bacillus pumilus KMM 62 culture liquid on different growth stages was isolated using ion-exchange chromatography. On the basis of pattern of specific chromogenic substrates hydrolysis and inhibitory analysis the protease was classified as subtilisin like serine protease. The molecular weight ofprotease is 31 kDa. Proteolytic activity towards Z-Ala-Ala-Leu-pNa substrate was maximal at pH 8-8.5. The optimal temperature for proteolytic activity was observed at a temperature of 30 degrees C, and the protein was stable within the pH range of 7.5-10.0. Bacillus pumilus KMM 62 subtilisin like serine protease was shown to have thrombolytic activity.     

A novel outer-membrane-associated protease in Escherichia coli.

Human gamma interferon produced by recombinant Escherichia coli was degraded by endogenous protease after cell disruption. Specific cleavages took place at the center of two pairs of basic amino acids (Lys-131-Arg-132 and Arg-142-Arg-143) in the C-terminal region, giving rise to products with molecular weights of 17,500 and 16,000. The proteolytic activity was associated with the outer membrane of E. coli. It was insensitive to the protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-L-lysine chloro-methyl ketone, EDTA, and p-chloromercuribenzoate. Benzamidine and the bivalent cations Zn2+ and Cu2+ inhibited the activity. Dynorphin A(1-13) (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys) was a good substrate and was preferentially cleaved at the center of Arg-6-Arg-7. Neither the amino nor carboxyl sides of Arg-9 and Lys-11 were digested. These results indicate that the protease specifically cleaves the peptide bond between consecutive basic residues and therefore is different from the known membrane enzymes, proteases IV, V, and VI. We have designated this new enzyme protease VII.     

Characterization of specific proteases associated with the surface of human skin fibroblasts, and their modulation in pathology.

Human skin fibroblasts were probed for cell surface protease activity. One activity removing dipeptides from the NH2-terminal end of Gly-Pro-pNA was specifically inhibited by di-isopropyl-fluorophosphate (DFP), phenylmethanesulphony fluoride (PMSF), and diprotin A, and thus was identified as dipeptidyl peptidase IV (DPP IV). A group of bestatin-sensitive N-exoaminopeptidase activities was also characterized when Ala-, Leu-, and Arg-pNA were used as chromogenic substrates. Using human monoclonal antibodies anti-CD 13 and anti-CD 26 that recognized, respectively, an N-Ala-aminopeptidase and DPP IV, it was found that human dermal fibroblasts expressed the CD 13 and CD 26 antigen on their surface. In addition, both peptidases were specifically immunoprecipitated by monoclonal antibodies anti-CD 13 and anti-CD 26 from plasma membranes. Cell surface proteolytic activities were also investigated in human fibroblasts derived from dermatological and rheumatic diseases (i.e., psoriasis, rheumatoid arthritis, and lichen planus). It was found that these fibroblasts also expressed both types of proteinases initially identified on normal skin fibroblasts and that the levels of Ala-aminopeptidase activities were similar in all cases. In contrast, the levels of Arg-, Leu-exoaminopeptidase, and DPP IV activities were significantly higher (up to 6.6-fold) in the three pathological fibroblast populations than in their normal counterparts. These proteolytic enzymes, therefore, can potentially serve as markers in dermatological diseases. Taken together, our results suggest that skin fibroblast-derived proteinases associated with both serine and N-aminopeptidase activities may play an important role by participating in the extracellular events associated with fibroblast behaviour.     

Maturation of isoprenylated proteins in Saccharomyces cerevisiae. Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro.

Eukaryotic polypeptides containing COOH-terminal-CXXX sequences can be posttranslationally modified by isoprenylation of the cysteine residue via a thioether linkage, proteolytic removal of the three terminal amino acids, and alpha-carboxyl methylation of the cysteine residue. Through the development of an indirect coupled assay, we have identified three in vitro activities in the yeast Saccharomyces cerevisiae that can catalyze the proteolytic cleavage of the three COOH-terminal amino acids of the synthetic peptide substrate N-acetyl-KSKTK[S-farnesyl-Cys]VIM. One of these is the vacuolar protease carboxypeptidase Y. Using a mutant strain deficient in this enzyme, we find evidence for an additional soluble activity as well as for a membrane-associated activity. These latter activities are candidates for roles in the physiological processing of isoprenylated protein precursors. They are both insensitive to inhibitors of serine and aspartyl proteinases but are sensitive to sulfhydryl reagents and 0.5 mM ZnCl2. The soluble activity appears to be a metalloenzyme, inhibitable by 2 mM o-phenanthroline but not by 1 mM N-ethylmaleimide, whereas the membrane-associated enzyme is inhibitable by 1 mM N-ethylmaleimide but not 2 mM o-phenanthroline. We show that the membrane-bound protease is not an activity of the membrane-bound methyltransferase, because protease activity is observed in membrane preparations that lack the STE14-encoded methyltransferase. The soluble activity appears to be a novel carboxypeptidase of approximately 110 kDa that catalyzes a processive removal of amino acids from the COOH terminus from both the farnesylated and non-farnesylated substrate, but not from three other unrelated peptides. Finally, we find no evidence for non-vacuolar membrane or soluble activities that catalyze the ester hydrolysis of N-acetyl-S-farnesyl-L-cysteine methyl ester.     

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Oligosaccharide structures of isolated human colonic mucin species

Purified human colonic mucin contains six distinct components which may be separated by DEAE-cellulose chromatography. Past studies defined the structure of oligosaccharide side chains from the most abundant species III, IV, and V which elute at intermediate salt concentrations. In these studies the structures of oligosaccharide side chains liberated from the remaining early and late eluting species I, II, and VI were determined after isolation by sequential conventional and high performance liquid chromatography through combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Mucin species I, II, and VI contained a less varied array of discrete oligosaccharide structures than that observed in the major mucin components. Mucin species I and II contained five and 10 structures, respectively, which account for 68 and 71% of total oligosaccharide content in these fractions. The predominant oligosaccharides of mucin species I included three neutral structures: a disaccharide GlcNAc beta (1-3)GalNAc-ol, a trisaccharide Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol, and a tetrasaccharide GlcNAc beta (1-4)Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol as well as two acidic components representing the sialylated forms of two of these oligosaccharides. Mucin species II contained these same oligosaccharides as well as four additional acidic structures, notably a disaccharide Neu alpha (2-6)GalNAc-ol and a hexasaccharide Gal beta (1-4)GlcNAc beta (1-3)Gal beta (1-4)GlcNAc beta (1-3) (NeuAc alpha (2-6))-GalNAc-ol, not identified in any other mucin species. The late eluting mucin species VI contained at least five discrete neutral oligosaccharides and six major acidic structures. While the majority of these structures had been previously isolated from the earlier eluting mucin species IV and V, species VI also contained di- and trisialylated oligosaccharides not identified in other mucin species. In conjunction with earlier studies of the major mucin species III, IV, and V, these data define the range of oligosaccharide structures present in human colonic mucin. These studies demonstrate that human colonic mucin possesses species with characteristic and distinguishable combinations of oligosaccharides which reflect variations of common core structures.     

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.     

Identification and evaluation as a DNA vaccine candidate of a virulence-associated serine protease from a pathogenic Vibrio parahaemolyticus isolate

A putative serine protease gene was cloned from the genomic DNA of Vibrio parahaemolyticus FYZ8621.4. The gene consisted of 1779 base pairs and encoded a 592 amino acid protein. The gene was expressed in Escherichia coli. The expressed protease was purified by Ni-NTA His-Bind Resin column and showed a 63 kDa band on SDS-PAGE. The protease exhibited proteolytic activity on gelatin agar plate and showed maximal proteolytic activity at pH 8.0 and 37 °C. It hydrolyzed N-α-benzoyl-L-tyrosine p-nitroanilide (BAPNA), but did not N-benzoyl-L-arginine ethylester (BAEE), N-benzoyl-L-tyrosine ethylester (BTEE) and N-acetyl-L-tyrosine ethylester (ATEE). Mutants at conserved residues Asp(51) (Asp(51)-Asn), His(89) (His(89)-Asp) and Ser(318) (Ser(318)-Leu, Ser(318)-Pro) lost proteolytic activities completely. The protein was confirmed to belong to serine protease. The purified serine protease was toxic to zebrafish with a LD(50) of 15.4 μg/fish. A DNA vaccine was constructed by inserting the mutated serine protease (Ser(318)-Pro) gene into pEGFP-N1 plasmid. The pEGFP-N1/m-vps was transfected in HeLa cells. The serine protease was confirmed to be expressed by fluorescence microscopy observation and Western blotting analysis. The pEGFP-N1/m-vps was further observed to express in muscle of the injected turbot (Scophthalmus maximus) by Western blotting seven days after immunization. Efficient protection against lethal V. parahaemolyticus challenge was observed on the vaccinated turbot with pEGFP-N1/m-vps, with the highest relative percent survival (RPS) of 96.11%. Significant specific antibody responses were also observed in the turbot vaccinated with the DNA vaccine. The results indicated that the serine protease might be a potential virulence factor and could be used as an efficient vaccine candidate for the disease control caused by V. parahaemolyticus.     

Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells

A proteinase was purified from crayfish haemocytes by affinity chromatography on heparin-sepharose and phenyl-sepharose, followed by DEAE-cellulose ion-exchange chromatography. This proteinase could mediate the conversion of prophenoloxidase (proPO) to its active form, phenoloxidase (PO), and its was therefore designated a prophenoloxidase activating enzyme, ppA.The purified ppA had a molecular mass of about 36,000 Da. Since ppA was a proteinase able to cleave chromogenic peptide substrates of trypsin, and serine proteinase inhibitors were strongly inhibitory towards ppA activity, the enzyme appeared to be a serine type proteinase. It exhibited maximal enzyme activity at neutral and slightly alkaline pH, and was sensitive to heat inactivation at 58°C.     

Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast

A previous study led to the discovery of new proteinases in yeast (Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch. Biochem. Biophys. 207, 445-454). The search for proteolytic enzymes active in the neutral pH range has been extended. Studies were done on a mutant lacking four well-known proteinases involved in protein degradation, the two endoproteinases A and B and the two carboxypeptidases Y and S. Twenty-nine chromogenic peptides (amino terminally blocked peptidyl-4-nitroanilides) as well as [3H]methylcasein were used as substrates in this search. For the detection of endoproteolytic activity using chromogenic peptide substrates two versions of the assay were used. In one system the direct cleavage of the 4-nitroanilide bond was measured. In the second, the cleavage of the chromogenic peptide at some site other than the 4-nitroanilide bond was measured. Both variations led to the discovery of multiple proteinase activities. Regulation of these proteolytic activities under different growth conditions of cells was observed. Proteolytic activity on [3H]methylcasein was also found. Ion-exchange chromatography and gel filtration were used for the reproducible separation of the multiple proteolytic activities.     

Biochemical characterization of the human complement protein C6. Association with alpha-thrombin-like enzyme and absence of serine protease activity in cytolytically active C6

Complement protein C6 has been proposed by others to be a serine protease whose activity is obligatory for complement-directed cell lysis. We separated the serine protease (Mr approximately 30,000) activity found associated with apparently homogeneous preparations of C6 from the hemolytically active C6 protein. The protease was characterized as thrombin-like based on substrate specificity, inhibitor profile, and kinetic studies. Although the proteolytic activity of C6 preparations was inhibitable by several inhibitors of serine proteases, the C6 hemolytic activity remained unaffected. Acid-induced (C(5,6)a complex formation between C5 and C6 (protease-free) was demonstrated by ion-exchange fast protein liquid chromatography, reversed-phase high performance liquid chromatography, and reactive cytolytic activity in the presence of C7, C8, and C9; but no cleavage of the alpha-chain of C5 was observed. Diisopropylphosphorofluoridate pretreatment of the components did not affect their ability to generate functionally active (C(5,6)a. Evidently, C6-associated thrombin is not required for formation of functional C(5,6)a. Thus, C6 does not function in the membrane attack pathway of complement as a serine protease. A method for the isolation of homogeneous C6 in the hemolytically fully active form is described. No free sulfhydryl group was detected in C6. The amino acid sequence of 20 amino-terminal residues was determined.     

Purification and partial characterization of a new proteolytic enzyme from the venom of Bothrops moojeni (CAISSACA).

A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.     

The purification and properties of cancer procoagulant from murine tumors.

The protease, cancer procoagulant, was isolated from three murine metastatic tumors and was purified to apparent homogeneity (SDS-PAGE) from Lewis lung cells by the sequence of (NH4)2SO4 precipitation, DE-53 anion-exchange chromatography, and Sephacryl 200 chromatography. The murine tumor enzyme has a molecular weight of 68,000 and Ca2+ is required for procoagulant and proteolytic activity; thus, the murine enzyme is very similar to that isolated from rabbit tumors. Two peptidyl chromogenic substrates of cancer procoagulant were discovered, facilitating kinetic and inhibition studies with the enzyme. The peptide substrate structures and the results of inhibition studies suggest that cancer procoagulant is thrombin-like in specificity but is a thiol protease.     

Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.     

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。     

Identification of oligopeptidase B in higher plants. Purification and characterization of oligopeptidase B from quiescent wheat embryo, Triticum aestivum

Proteolytic enzymes in general, and cysteine proteases in particular, play key roles in seed germination and early seedling growth. However, the precise mechanism by which the serine proteases are regulated remains unclear. Trypsin-like activity was detected in wheat germ (quiescent embryo) and this activity increased in the germinating embryo. In this work, a trypsin-like serine protease expressed in wheat germ was purified to homogeneity by chromatography through DEAE-cellulose, phenyl-Sepharose, Ultrogel AcA-34 and Blue-Sepharose. The molecular mass of the enzyme was estimated to be 81 kDa by SDS-PAGE under reducing conditions. Amino acid analysis of the peptides generated following digestion of the enzyme with lysyl endopeptidase indicated that the enzyme is a plant homologue of Escherichia. coli oligopeptidase B. The subsite specificity of the enzymes differ, although both enzymes hydrolyze synthetic substrates and model peptides at the carboxyl side of basic amino acids. The wheat enzyme is more sensitive to leupeptin and antipain than the E. coli emzyme. These results provide the basis for characterizing plant oligopeptidase B and contribute to our understanding of its role in the early development of seedlings.     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.