Unusual catalytic activities and functions of cholinesterases.

Cholinesterases (acetylcholinesterase and butyrylcholinesterase) have been shown to exhibit not only esterase activity but also an amine sensitive aryl acylamidase and a metallo-carboxypeptidase activities. There is also evidence to indicate that they have functions in the substantia nigra of brain, in neural cell differentiation, cell division and tumorigenesis, cell-adhesion and detoxication mechanisms. Butyrylcholinesterase is suggested to act as a back-up enzyme in acetylcholinesterase knock-out mice. Cholinesterases have catalytic or non-catalytic roles in these functions. Partial sequence homology to many other proteins having different functions and a metal binding site which can influence functions are probably factors that confer the non-cholinergic functions and activities on cholinesterases.

Amyloid beta peptide processing, insulin degrading enzyme, and butyrylcholinesterase.

Amyloid beta peptide implicated in Alzheimers disease is cleaved by insulin degrading enzyme (IDE). Abnormal cholinesterases similar to butyrylcholinesterase (BChE) are found in Alzheimer brain. The similarities between IDE and BChE (which is known to have an arylacylamidase and a metallocarboxypeptidase-like activity) such as their zinc metalloenzyme nature, their localization in glia and their ability to bind amyloid peptide in Alzheimers disease raise interesting questions.

Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme.

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.

Selective inactivation of butyrylcholinesterase with metal chelators suggests there is more than one metal binding site.

Cholinesterases exhibit functions apart from their esterase activity. We have demonstrated an aryl acylamidase and a zinc stimulated metallocarboxypeptidase activity in human serum butyrylcholinesterase. To establish the presence of zinc binding sites in the enzyme we examined the effect of metal chelators on its catalytic activities. The metal chelators 1,10-phenanthroline and N,N,N',N'-tetrakis (2-pyridyl methyl)ethylene diamine (TPEN) inhibited all the three catalytic activities in the enzyme. However, EDTA inhibited the peptidase activity exclusively without affecting the cholinesterase and aryl acylamidase activities. The catalytic activities were recovered upon removal of the chelator by Sephadex G-25 chromatography. Pre-treatment of the enzyme with any one of the three chelators resulted in the binding of the enzyme to a zinc-Sepharose column or to 65Zn2+. Histidine modification of the enzyme pretreated with chelators resulted in abolition of 65Zn2+ binding and zinc-Sepharose binding. Whereas the binding studies demonstrated removal of a metal from a Zn2+ binding site, attempts to remove the metal responsible for catalytic activity were unsuccessful. Atomic absorption spectroscopy indicated approximately 2.5 mol of zinc per mol of enzyme before treatment with EDTA and 1 mol zinc per mol enzyme after EDTA treatment. The results indicate that there are at least two metal binding sites on butyrycholinesterase. The presence of two HXXE...H sequences in butyrylcholinesterase supports these findings. Our studies implicate a zinc dependent metallocarboxypeptidase activity in the non-cholinergic functions of butyrylcholinesterase.

Mycobacterium leprae binds to a 25-kDa phosphorylated glycoprotein of human peripheral nerve.

Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease. Little is known about the bacteria-nerve protein interaction. We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve. This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity. This M leprae-protein interaction could be of importance in the pathogenesis of leprosy.

M. leprae binds to a 28-30-kDa phosphorylated glycoprotein of rat peripheral nerve.

To understand Mycobacterium leprae-peripheral nerve interaction, we have investigated the binding of M. leprae to rat peripheral nerve proteins in an in vitro model using 32P-phosphorylated proteins of the peripheral nerve. Intact M. leprae binds to a major phosphorylated protein of 28-30 kDa and, to a minor extent, to a few proteins of molecular weight 45-55 kDa. This binding was more specific for M. leprae since only insignificant binding was observed with other bacteria, such as M. bovis or Escherichia coli. M. leprae did not show binding to several phosphorylated proteins of the rat brain. The 28-30-kDa binding protein of the rat peripheral nerve was found to be a glycoprotein by concanavalin A-Sepharose column chromatography.

Protein phosphorylation in human peripheral nerve: altered phosphorylation of a 25-kDa glycoprotein in leprosy.

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg2+ or Mn2+ was essential for maximum phosphorylation although Zn2+, Co2+, and Ca2+ could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.

Evidence for a Zn(2+)-binding site in human serum butyrylcholinesterase.

Purified human serum butyrylcholinesterase after treatment with either of the metal chelators EDTA or NaCN was able to bind to a Zn(2+)-chelate-Sepharose affinity column and was eluted from the column by EDTA or imidazole. Prior EDTA treatment of the enzyme was essential for binding to this affinity column. The enzyme could be labelled with (65)Zn(2+) after EDTA treatment of the enzyme. Diethylpyrocarbonate modification of histidine residues in the EDTA-treated enzyme resulted in the abolition of both binding to the Zn(2+)-chelate-Sepharose column and labelling by (65)Zn(2+). Stoicheiometry of (65)Zn(2+) binding indicated approximately 0.85 mol of Zn(2+)/mol of subunit of the EDTA-treated enzyme. EDTA or NaCN treatment resulted in the loss of thermal stability of the enzyme at 37 degrees C which could not be reversed by Zn(2+). Whereas the cholinesterase activity of butyrlcholinesterase was not affected by EDTA, there was significant loss of its carboxypeptidase activity in the presence of EDTA, and the loss could be reversed by added ZnCl2. These results suggest the presence of a Zn(2+)-binding site on human serum butyrylcholinesterase and the involvement of histidine residues in the metal binding. The presence in human serum butyrylcholinesterase of a sequence HXXE...H found in many known Zn(2+)-containing enzymes supports these findings.

Phosphorylation of casein, fibrinogen and calmodulin by a glycoprotein protein kinase from monkey cerebellum: a casein kinase II-like enzyme.

A glycoprotein protein kinase was isolated from monkey cerebellum by polylysine-Sepharose chromatography and affinity chromatography on Sepharose 4B coupled to the lectin, Concanavalin A. The protein kinase phosphorylated casein on serine and threonine residues and was stimulated by polylysine, polyarginine, spermine, histone, protamine and sphingosine, but was inhibited by heparin, poly (Glu, Ala, Tyr) and poly (Glu, Tyr). These characteristics were typical of casein kinase II. The protein kinase also phosphorylated fibrinogen and calmodulin and exhibited similar characteristics of stimulation by polylysine or polyarginine. The phosphorylation of fibrinogen (a glycoprotein), but not casein or calmodulin (non-glycoproteins), was significantly inhibited by Concanavalin A. Unlike casein kinase II, the enzyme did not undergo autophosphorylation. The collective results suggested that the enzyme from monkey cerebellum was a casein kinase II-like protein kinase and that phosphorylation of a glycoprotein substrate (fibrinogen) by the kinase could be influenced by a carbohydrate binding lectin.

Noncholinergic functions of cholinesterases.

Cholinesterases (acetylcholinesterase and butyrylcholinesterase) exhibit additional catalytic activities apart from their well-known action in hydrolyzing choline esters. An amine-sensitive aryl acylamidase activity is exhibited by both acetyl- and butyrylcholinesterases. A metallocarboxypeptidase-like activity is found associated with both acetyl- and butyrylcholinesterases. The peptidase activity exhibited by butyrylcholinesterase was located in a 50-kDa COOH-terminal fragment. Acetylcholinesterase is implicated in noncholinergic functions in the substantia nigra. A relationship between tumorigenesis, cell differentiation, and cholinesterases has been speculated. The sequence similarities between different esterases, lipases, thyroglobulin, cell adhesion proteins, and cholinesterases would make it appear that cholinesterases are capable of exhibiting more than one biological activity and their functions are wider than what is hitherto known.

Sulfation of proteins in the primate cerebellum and young rat brain.

Protein tyrosine sulfotransferase activity in a 20,000 g sedimentable fraction of monkey cerebellum was demonstrated. Both endogenous proteins and the exogenous substrate poly (Glu, Ala, Tyr) random copolymer were sulfated. The copolymer in the low molecular mass range (approx 20 kDa) was preferentially sulfated. Addition of copolymer inhibited sulfation of endogenous proteins. Mg2+ and Mn2+ promoted sulfation. 35S-Labeled proteins from monkey cerebellum and young (10 days old) rat brain were subjected to lectin-Sepharose chromatography to identify the presence of sulfated glyco-proteins. Labeled proteins from both these sources could bind and get eluted from Concanavalin A-Sepharose and Ricinus Communis agglutinin-Sepharose column suggesting the presence of mannose or galactose containing glycosulfoproteins.

Stimulation by lysosomal enzymes and mannose-6-phosphate of a phosphoprotein phosphatase activity associated with the lysosomal enzyme binding receptor protein from monkey brain.

Previously we have isolated a lysosomal enzyme binding receptor protein from monkey brain that exhibits protein kinase activity and undergoes phosphorylation on serine and tyrosine residues. Using the 32P-labelled receptor protein, we have found that the lysosomal enzyme fucosidase and mannose-6-phosphate, which are ligands for the receptor, stimulated a protein phosphatase activity associated with the receptor protein. Stimulation of protein phosphatase activity using the 32P-labelled receptor protein was demonstrated both by the loss in radioactivity of the receptor and by the release of 32P-phosphate. There was no stimulation by a non-lysosomal glycoprotein enzyme, or by the sugars mannose or glucose. Both serine-phosphate and tyrosine-phosphate residues were dephosphorylated. Stimulation of protein phosphatase activity by fucosidase and mannose-6-phosphate was also demonstrated using as substrate histone 32P-labelled, on serine/threonine or tyrosine residues. Insulin-like growth factor II, another known ligand for the lysosomal enzyme binding receptor, did not show any significant effect, either on the phosphorylation or dephosphorylation of the receptor protein. Our previous and present results suggest that a phosphorylation/dephosphorylation mechanism may be operative in the ligand binding and functions of the receptor.

The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase.

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.