Degradation of myelin basic protein in myelin by protease in cerebrospinal fluid and effects of protease inhibitors

Neutral protease is shown to be present in cell-free human cerebrospinal fluid. Incubation of heated human myelin with CSF at 25°C resulted in a marked reduction of myelin basic protein (MBP) with time. Degradation products appeared at apparent mol wt 14 KDa and 12 KDa on polyacrylamide gel electrophoresis. Optimal pH of the protease was 7.0. This protease was activated by calcium ion. Degradation of MBP was inhibited by FOY305 (camostat mesilate), Trasylol^®, and Leupeptin, but not a specific calcium-activated neutral protease inhibitor, E-64-a. FOY305, which is a synthesized specific serine protease inhibitor, was the strongest inhibitor of all. The role of this protease in CSF has not been elucidated. In may be related to the physiological turnover of MBP, and may affect myelin maintenance in pathological conditions such as demyelination.     

Stimulative Effect of a Casein Hydrolysate on Exocrine Pancreatic Secretion That is Independent of Luminal Trypsin Inhibitory Activity in Rats

We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 μg/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 μg/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.     

Stimulative effect of a casein hydrolysate on exocrine pancreatic secretion that is independent of luminal trypsin inhibitory activity in rats.

We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 micrograms/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 micrograms/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.     

Neutral protease activity in lymphocytes of lewis rats with acute experimental allergic encephalomyelitis

Lymphocytes from popliteal and inguinal lymph nodes of Lewis rats with acute EAE as a result of injection of lyophilized guinea pig myelin in Freund's complete adjuvant exerted strong proteolytic activity at neutral pH toward myelin basic protein. After injection of myelin the level of proteolytic activity remained about the same as that in lymphocytes from Freund's adjuvant-injected controls until about day 10 after injection, just before the onset of paralytic symptoms; then the proteolytic activity increased to approximately double its former level. Myelin basic protein was hydrolyzed by whole lymphocytes, but more activity was unmasked by homogenization. Similar results were also obtained using lymphocytes from thymus of EAE and control animals. Lymphocytes with high levels of proteolytic activity were not absorbed by glass wool, did not stain with neutral red, nor did they phagocytose antibody-coated sheep red blood cells. Thymus and lymph node lymphocytes cleaved myelin basic protein to three major peptides and a fourth minor peptide, while spleen lymphocytes hydrolyzed basic protein at only one point resulting in two peptides whose molecular weights added up to that of myelin basic protein. The protease activity was inhibited by 5×10^–3 Mp-chloromercuribenzoate and by phenylmethyl sulfonyl fluoride, TPCK, and soybean trypsin inhibitor, therefore the enzymatic activity probably depends on a serine residue and a sulfhydryl group. The bulk of the enzymatic activity is mostly membrane bound with the highest specific activity and total activity contained in a lysosomal-mitochondrial fraction. In view of the infiltration of lymphocytes into the brain substance in acute EAE, it is suggested that these cells may contribute to the destruction of myelin which is usually attributed to the monocyte or macrophage.     

A lymph node neutral proteinase acting on myelin basic protein

A neutral protease present in inguinal and popliteal lymph nodes of rats with acute experimental allergic encephalomyelitis (EAE), rats injected with Freund's adjuvant, and rats that are normal has been found to hydrolyze basic protein present in purified brain and spinal cord myelin. The enzyme has been enriched by ammonium sulfate precipitation, and its properties have been studied. The protease activity toward different substrates was very specific and decreased in the following order: Protamine sulfate = polylysine (MW 183,000) > myelin basic protein > histone > polylysine (MW 2000) > polyarginine > cytochrome c. Other proteins including casein, freshly denatured hemoglobin, egg albumin, bovine serum albumin, and ribonuclease were ineffective as substrates. The pH curve showed a peak at pH7 for rat myelin, isolated beef basic protein, and histone. A possible role for this enzyme in demyelination in acute experimental allergic encephalomyelitis is suggested.     

Partial purification and characterization of neutral proteases in lymph nodes of rats with experimental allergic encephalomyelitis

Two kinds of neutral protease activities in lymph nodes from Lewis rats with acute experimental allergic encephalomyelitis (EAE) have been separated and partially purified and characterized. A soluble enzyme preparation enriched by gel filtration and ion-exchange chromatography hydrolyzes myelin basic protein, polylysine, and other basic proteins with an optimum pH at 6.0–6.5. It is inhibited byp-chloromercuribenzoate, and thus appears to be a mixture of thiol proteases. Another fraction containing proteolytic enzyme activity is strongly bound to the insoluble lymph node residue, and it also hydrolyzes myelin basic protein and histone, but not polylysine. It has a pH optimum above 7.5, is inhibited by phenylmethylsulfonyl fluoride, thus resembling elastase, but does not hydrolyze elastin-Congo red. The insoluble enzyme preparation hydrolyzes basic protein to 4–5 peptides in a pattern on polyacrylamide gels resembling that of the hydrolysis of basic protein by whole lymphocytes; the soluble enzyme mixture produces small fragments not retained on gels. Lymphocytes are a major component of the cells inflitrating the nervous system in experimental allergic encephalomyelitis, and neutral proteases contained in these cells may contribute to the degradation of myelin, especially of the basic protein.     

Characterization of Myelin-Associated Glycoprotein (MAG) Proteolysis in the Human Central Nervous System

Purified human central nervous system myelin contains an endogenous cysteine protease which degrades the 100-kDa myelin-associated glycoprotein into a slightly smaller 90-kDa derivative called dMAG, and which has been implicated in demyelinating diseases. The native proteolytic site in human MAG was determined in order to characterize this cysteine protease in humans further. This was accomplished by identifying the carboxy-terminus of purified dMAG. The results of these experiments, in conjunction with peptidolysis assays of myelin, demonstrated that the enzyme which proteolyses MAG is extracellular and has cathepsin L-like specificity. Furthermore, it was shown that this cathepsin L-like activity potentially was regulated by the endogenous extracellular inhibitor cystatin C.     

Observations on the effects of protease inhibitors on the suppression of experimental allergic encephalomyelitis

Inhibitors of proteolytic enzymes were tested for their ability to suppress the clinical signs and CNS lesions produced by injection of purified myelin in complete Freund's adjuvant into Lewis rats. Pepstatin or a series of neutral protease inhibitors including aprotinin, soybean trypsin inhibitor, leupeptin, antipain, transaminomethyl cyclohexane carboxylic acid (AMCA), -amino caproic acid (EACA) nitrophenyl guanidino benzoate (NPGB),d- andl-polylysine, or a new commercial protease inhibitor, dipropionyl Rhein (DPR) were injected daily beginning on day 7 after immunization of rats with myelin. Aprotinin and soybean trypsin inhibitor exacerbated the symptoms and lesions of experimental allergic encephalomyelitis (EAE), leupeptin and antipain had no effect, and the plasminogen activators AMCA, EACA, NPGB, as well as poly-l- and poly-d-lysine and DPR suppressed various aspects of EAE. The measurement of acid protease as a biochemical method for quantitation of the degree of cellular infiltration into the CNS is proposed, and the results with the various treatments presented. AMCA and NPGB may exert their effects at the site of entrance of the lymphoid cells into the CNS.     

Mushroom (Agaricus bisporus) compost quality factors for predicting potential yield of fruiting bodies

A quality model has been developed from parameters determining the interactions of physical, chemical, and biological factors during the preparation of mushroom compost for growing Agaricus bisporus. Our results show that a partial least square model based on the combination of pH, dry matter, ammonia, carbon, hydrogen, ash, Cu, Fe, and Na could explain nearly 90% of the variation in mushroom yield obtained from four compost comparative trials. The yields in the data base for generating the model ranged from 138 to 305 kg per ton of compost. The validity of the yield model has been confirmed in a trial carried out in collaboration with experienced commercial growers. This has significant implications for compost producers, as production efficiencies can be maintained by targeting the important parameters.     

Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth

A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte-myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.     

Poster Sessions CP12: Neurotrauma & Injury. Characterization of cathepsin B expression following contusion spinal cord injury (SCI)

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI‐induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real‐time PCR and immunoblots, respectively. Both the contusion injured animals and the time‐matched sham controls exhibited elevated pro‐enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post‐SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post‐SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real‐time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up‐regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris

Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.     

Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (pepstatin)

Intraperitoneal administration of pepstatin (2 mg/day for 5 weeks) to Lewis rats subjected to experimental allergic encephalomyelitis (EAE) (induced by guinea pig spinal cord and pertussis vaccine) suppressed the appearance of clinical signs of disease, and reduced the severity and incidence of CNS lesions normally associated with this disease. Administration of pepstatin for shorter periods to Lewis rats, or BSVS mice, or guinea pigs challenged with myelin basic protein delayed, but did not prevent clinical signs of EAE, but was accompanied in all cases by a less severe histopathology.     

A peptide inhibitor of HIV-1 protease using alpha, beta- dehydro residues: a structure based computer model

HIV-1 encodes an aspartic protease, an enzyme crucial to viral maturation and infectivity. It is responsible for the cleavage of various protein precursors into viral proteins. Inhibition of this enzyme prevents the formation of mature, infective viral particles and therefore, it is a potential target for therapeutic intervention following infection. Several drugs that inhibit the action of this enzyme have been discovered. These include peptidomimetic inhibitors such as ABT-538 and saquinavir, and structure based inhibitors such as indinavir and nelfinavir. Several of these have been tested in human clinical trials and have demonstrated significant reduction in viral load. However, most of them have been found to be of limited clinical utility because of their poor pharmacological properties and also because the viral protease becomes rapidly resistant to these drugs on account of mutations in the enzyme. One way to overcome these limitations is to design an inhibitor that interacts mainly with the conserved residues of HIV-1 protease. By a rational drug design approach based on the high resolution X-ray crystal structure of the HIV-1 protease with--MVT 101 (a substrate based inhibitor) and the specific design principles of peptides containing dehydro-Alanine (delta Ala) derived from our earlier studies, we have designed a tetrapeptide with the sequence: NH2-Thr-delta Ala-delta Ala-Gln-COOH. Energy minimization and molecular modelling of the interaction of the designed tetrapeptide with the inhibitor binding site indicate that the inhibitor is in an extended conformation and makes excessive contacts with the viral enzyme at the interface between the protein subunits. The designed inhibitor has 33% of its interaction with the conserved region of HIV-1 protease which is of the same order as that of MVT 101 with the enzyme.     

Degradation of myelin proteins by cathepsin B and inhibition by E-64 analogue

Purified human brain myelin was isolated, heat-treated to inactivate the endogenous proteolytic activity and incubated with cathepsin B purified from rat liver, at pH 6.0. Incubation resulted in a marked reduction of myelin basic protein (BP) and partial breakdown of proteolipid protein or Wolfgram protein. Degradation of myelin proteins was inhibited by E-64 analogue (E-64-a). E-64 is a specific thiol protease inhibitor isolated from a solid culture of Aspergillus japonicus. The present study suggests that cathepsin B may play some role in demyelination.     

Crystal structure of human epidermal kallikrein 7 (hK7) synthesized directly in its native state in E. coli: insights into the atomic basis of its inhibition by LEKTI domain 6 (LD6)

Human kallikrein 7, a major protease of human skin, has been synthesized directly in its native conformation in Escherichia coli by forcing the secretion of the newly synthesized polypeptide into the bacterial periplasm. The procedure yields a stable kallikrein 7 with highly specific activity that is inhibited efficiently by its specific inhibitor LEKTI domain 6. The protein was crystallized, and its three-dimensional structure was solved in the absence of protease inhibitors. The structure obtained agrees with that reported recently for human tissue kallikrein 7 crystallized in the presence of protease inhibitors from a preparation obtained in a baculovirus protein expression system. A model of the interaction between the protease and its inhibitor is proposed on the basis of both the three-dimensional structure of human tissue kallikrein 7 reported here and that of the LEKTI domain 6 solved previously by NMR.     

Presence of the Plasma Membrane Proteolipid (Plasmolipin) in Myelin

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.     

Effect of protease inhibitors on yield of HSV-1-based viral vectors

The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors.     

Limited Digestion of Guinea Pig Myelin Basic Protein and Its Carboxy-Terminal Fragment (Residues 89–169) with Staphylococcus aureus V8 Protease

Staphylococcus aureus V8 protease has been reported to have a strict specificity for cleavage of the Glu-X bond in ammonium bicarbonate (pH 7.9). With myelin basic protein and one of its major peptic fragments (residues 89-169) as substrates, selective cleavage of Asp(32)-Thr(33), Asp(37)-Ser(38), and Glu(118-Gly(119) bonds was observed, as well as the unusual cleavage of the Gly(127)-Gly(128) bond. The Asp-Glu and Glu-Asn bonds in the sequence of Gln-Asp-Glu-Asn-Pro(81-84) were resistant to V8 protease attack. The following peptides were identified as products of limited cleavage of basic protein by V8 protease: (1-32), (1-37), (33-169), (38-169), (33-118), (38-118), (33-127), (38-127), (119-169), and (128-169). Cleavage of the peptic peptide (89-169) yielded fragments (89-118), (89-127), (119-169), and (128-169). All peptides were identified by amino acid analysis, as well as NH2- and COOH-terminal analyses. Time course studies with basic protein showed that V8 protease initially attacked the bonds between Asp(32) and Thr(33) and Asp(37) and Ser(38). With peptide (89-169) the initial cleavage was between Glu(118) and Gly(119). Peptides (89-118) and (89-127) were encephalitogenic in the Lewis rat. The activity of these peptides in the rat confirms the presence of a minor encephalitogenic site in guinea pig basic protein. Peptide (89-127) was encephalitogenic in the guinea pig, as expected, because it contains the intact encephalitogenic site. V8 protease digestion of basic protein yields some interesting new fragments, not previously available for biologic studies.     

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa (Medicago sativa)

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.