Biotinylation of biotinidase following incubation with biocytin

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Clinica Chimica Acta 233 (1995) 39-45

Biotinylation of biotinidase following incubation with biocytin Jeanne Hymes, Kristin Fleischhauer, Barry Wolf* Departments of Human Genetics and Pediatrics, Medical College of Virginia~Virginia Commonwealth University, Richmond, VA 23298, USA

Received 5 April 1994; revision received 6 August 1994; accepted 8 August 1994

Abstract Human serum biotinidase, purified to homogeneity (1920 units/rag protein), was incubated with biocytin prior to electrophoresis and transblotting with avidin-peroxidase. Avidin reacted with biotinidase maximally when incubated at pH 7.5-9, less at pH 7 and none below pH 7. No avidin reactivity occurred when biotinidase was incubated with biotin or in the absence of biocytin. Inclusion of the nucleophilic acceptors, ethanolamine or hydroxylamine, to the incubation mixture with biocytin and biotinidase resulted in loss of avidin reactivity. High concentrations of mercaptoethanol also prevented avidin reactivity. These results suggest that biotinidase can be biotinylated in the presence of biocytin at neutral to alkaline pH probably through a thioester bond formed with a cysteine residue in the active site of the enzyme. Biotinidase may then function as a biotinylating enzyme when incubated with appropriate nucleophilic acceptors. Keywords: Biotinidase; Biotinylation; Biocytin; Biotin; Avidin; Thioester

1. Introduction Biotin is the coenzyme for four mammalian carboxylases Ill. These carboxylases play important roles in fatty acid synthesis, gluconeogenesis and the catabolism o f specific branched-chain amino acids. Biotinidase recycles biotin by cleaving biocytin derived from the proteolytic degradation o f the biotin-dependent carboxylases [2]. * Corresponding author, Department of Human Genetics, Medical College of Virginia, PO Box 33, MCV Station, Richmond, VA 23298, USA. Tel: (804) 786-9632; Fax: (804) 786-3760. 0009-8981/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0009-898 ! (94)05965-U

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J. Hymes et al./ Clin. Chim. Acta 233 (1995) 39-45

The hydrolysis of biocytin to biotin and lysine occurs at pH 4-6 with little or no hydrolysis at physiologic or alkaline pH [3]. Biotinidase deficiency, an autosomal recessively inherited disorder, is characterized by neurologic and cutaneous abnormalities [4]. Treatment with pharmacological doses of biotin can prevent or eliminate many of the adverse features of the disorder. In profound biotinidase deficiency, plasma biotin concentrations are frequently low and renal excretion of biotin is elevated [5]. These findings suggested that biotinidase may act as a biotin-carrier protein [2]. We propose that biotinidase may be a major biotin-binding protein in serum (pH 7.4) and that biotin binding has two components. One is the formation of biotinyl-acyl-biotinidase that is stable at physiologic pH and the other is the transferring of biotin from biotinyl-biotinidase to various, possibly specific, nucleophilic acceptors. Biotinylation of biotinidase and transferring of biotin is facilitated by biocytin, not free biotin. 2. Materials and methods

2.1. Monoclonal antibodies to biotinidase Monoclonal antibodies were produced (Massey Cancer Center Hybridoma/ Monoclonal Laboratory, VA Commonwealth University, Richmond, VA) to human serum biotinidase that was purified to homogeneity (22 000-fold) as described previously [6]. An IgG fraction of the immune ascites fluid was prepared by ammonium sulfate precipitation [7]. 2.2. Incubation of purified biotinidase with biocytin and biotin Purified biotinidase (8.7/~g/ml) was incubated in 0.1 M buffer at various pHs (sodium citrate at pH 3, sodium acetate at pH 4 and 5 and sodium phosphate at pH 7, 8 and 9). Biotin or biocytin was added at a final concentration of 0.2 mM and the reaction mixture (total volume 0.020 ml) was incubated for 30 min at 37°C. The reaction was stopped by freezing until the samples were electrophoresed. Sample buffer containing sodium dodecyl sulfate (SDS), without mercaptoethanol, was added prior to SDS-PAGE. Twenty microliters of the mixture (0.17/~g of biotinidase) were added per gel well when developed with avidin and 7 #1 (0.06 #g) were added when developed with antibody. 2.3. Electrophoresis Native 10% polyacrylamide tricine minigels and 12% SDS-polyacrylamide tricine minigels (Millipore, Bedford, MA) vertical electrophoresis (Hoeffer, San Francisco, CA) were performed according to the manufacturer's directions, except that mercaptoethanol was eliminated from the SDS-PAGE sample buffer. 2.4. Blotting procedures Following electrophoresis proteins were transferred to nitrocellulose and blotted as described previously [8]. When avidin was used the milk was filtered through Whatman No. 1 filter paper. Avidin transblots. After washing the blocked nitrocellulose three times with Tris buffered saline (pH 7.5) containing 0.1% Tween 20 (TBST) for 5 min each, avidin-

J. Hymes et aL / Clin. Chim. Acta 233 (1995) 39-45

41

peroxidase complex (Sigma, St. Louis, MO), a reagent used to detect biotinylated proteins, diluted 1:15 000 in TBST, was added and reacted with the membrane for 30 min at 25°C. After three TBST washes the membrane was developed for 1 min with enhanced chemiluminescence (ECL) peroxidase substrate (Amersham, Arlington Heights, IL), placed between clear plastic sheets and exposed to X-ray film. Anti-biotinidase immunoblots: The immunoblot was prepared as described above. IgG monoclonal antibody to biotinidase, diluted 1:1200 in TBST containing 1% BSA, was then incubated with the membrane at 4°C for 3 h. The remaining steps were as previously described except anti-mouse IgG and mouse peroxidase antiperoxidase (PAP) antibodies were used instead of anti-rabbit IgG and rabbit PAP antibodies [8]. These blots were then developed by ECL as above. 3. Remits

Human serum biotinidase, purified to homogeneity (22 000-fold), was incubated at various pH values with biocytin, biotin or buffer and then electrophoresed on separate, but identical, native or SDS tricine polyacrylamide gels. The blot of native gel electrophoresis (Fig. 1) shows that avidin reacted with biotinidase only when incubated with biocytin. No avidin reactivity was evident when biotinidase was incubated with biotin or buffer. Avidin reacted with biotinidase that had been incubated with biocytin at pH 7.5-9. Avidin reactivity was detected at pH 7 only after longer film exposures (not shown) and was undetectable below pH 7. When biotinidase was incubated with buffer, biocytin or biotin as above and SDSPAGE was performed (Fig. 2A) avidin reactivity occurred only when biotinidase,

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Fig. 1. Avidin reactivity of biotinidase on immunoblots of native gel. Human serum biotinidasc puri~ed to homogeneity was incubated with buffer (N), biocytin (B'), or biotin (B) at pH 3-9 as described in Methods and then electrophoresed on native tricinc polyacrylamide gels. The protein was transferred to nitrocellulose and then allowed to react with avidin-peroxidase and detected by E C L

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Fig. 2. Avidin reactivity of biotinidase on immunobiots of SDS gel. Human serum biotinidase was incubated at pH 3 - 9 with buffer (N), biocytin (B'), or biotin (B) and then electrophoresed on SDS polyacrylamide gels as described in Methods. The protein was transferred to nitrocellulose, reacted with avidinperoxidase and developed by ECL. (B) Monoclonal anti-biotinidase detection of biotinidase, 78 kDa, on immunoblots of SDS gel. Duplicate SDS-PAGE blots, as shown in (A), were reacted with monoclonal antibody to biotinidase and developed by ECL as described.

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J. Hymes et al. / Clin. Chim. .4cta 233 (1995) 39-45

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Fig. 3. Effectof addition of nucleophilicreagents on avidin reactivityon an immunoblot of SDS polyacrylamidetricine gel. Lane !: biotinidasewas incubated with biocytinat pH 9 as describedin methods. Lane 2: same as lane 1, except that 0.1 M hydroxylamine,adjusted to pH 9 with NaOH, was included in the reaction mixture. Lane 3: same as lane 1, exceptthat 0.1 M ethanolamine, adjusted to pH 9 with HCI, was added to the reaction mixture. Lane 4: biotinidase was incubated in buffer without biocytin.

78 kDa, was incubated with biocytin and the reactivity was similarly pH-dependent. Results of immunoblots using anti-biotinidase indicated equal quantities of the enzyme protein in all samples (Fig. 2B). When the nucleophilic reagents, ethanolamine and hydroxylamine (0.1 M), were added to the incubation mixtures prior to SDS-PAGE, avidin reactivity was eliminated (Fig. 3). Avidin reactivity was also diminished when sample buffer containing mercaptoethanol (1.4 M) was added to the reaction mixture before SDSPAGE (data not shown). Therefore mercaptoethanol was omitted from the sample buffer and the samples were not boiled.

4. Discussion Biotinidase occurs ubiquitously in animal tissues and large quantities of the enzyme are found in serum [9]. The enzyme's natural substrate, biocytin, is cleaved optimally at pH 4-6, whereas serum pH is 7.4 [3]. The results of studies of biotin binding to biotinidase have been equivocal. One study examined the non-covalent binding of radioactive biotin to purified biotinidase and serum and determined that biotinidase was the only protein in serum that exchanged labelled biotin [10]. Another study using labelled biotin indicated that most of the biotin in plasma was not bound to protein [11]. This is the first time biocytin has been used to study biotinidase as a covalent biotin-binding protein. Studies of biotinidase that have examined biotinidase as a non-covalent biotin-binding protein were performed using radioactively labeled biotin in equilibrium with the enzyme. Radioactive biocytin could not be made with sufficiently high specific activity to perform similar binding studies [11]. The ECL technique used in the present study is as sensitive as the radiolabeling methodologies [121. Avidin reacted with biotinidase after incubation with biocytin, but not after incubation with biotin or buffer. Since biotinylated biotinidase was present after elec-

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J. Hymes et al. / Clin. Chim. Acta 233 (1995) 39-45

trophoresis, blotting and extensive washings of the membrane biotin binding appears to be covalent. The biotinylated biotinidase is stable at neutral to alkaline pH, except in the presence of high concentrations of mercaptoethanol or upon addition of the nucleophilic reagents hydroxylamine or ethanolamine. Each of these reagents cleaves thioesters [13]. That a cysteine residue is in or near the active site of biotinidase is supported by studies of sulfhydryl inhibitors [9,14]. Because mercaptoethanol at high concentration prevents avidin reactivity, the binding of biotin to the enzyme is likely through a thioester moiety formed during cleavage of biocytin. Biotinidase has been shown to have I 0-fold higher activity in the presence of mercaptoethanol than in its absence and mercaptoethanol added to serum increased biotinidase activity as much as 4fold [14]. Since thioesters are sensitive to mercaptoethanol this activation may be caused by increased turnover of biotinyl-biotinidase. Ethanolamine and hydroxylamine interfere with the biotinylation of biotinidase, probably by nucleophilic attack of the biotinyl-thioester intermediate of biotinidase. Formation of biotin-hydroxamate has been shown to occur when biotinidase is incubated with biotinyl-p-aminobenzoate, an artificial substrate of biotinidase, and hydroxylamine at pH 7.2 [9]. In these studies enzyme activity was not inhibited with as high as 1.44 M hydroxylamine. In fact, after 30 min of incubation, twice as much p-aminobenzoate was released from biotinyl-p-aminobenzoate by the enzyme in the presence of this concentration of hydroxylamine. In a recent study, biocytin, but not biotin, injected into brain or lateral ventricles of rats result in selective labeling of particular subpopulations of central nervous system neurons [15]. In these studies the biotinyl moieties in the coronal sections were identified by their reactivity with avidin. These biotinyl compounds were present after extensive perfusion of the animal following injection of biocytin and after thorough washing of the tissues in preparation for visualization with avidin-peroxidase. Although the mechanism for this biotinylation is not known, the results indicate that biotin must be covalently attached to specific components of brain or they would be lost during the perfusion or autoradiographic preparation. Biotinidase may play a role in the biotinylation of these tissues. Because neurological problems usually occur in untreated individuals with biotinidase deficiency before indications of carboxylase deficiency [16], biotinylation of biotinidase and/or other molecules in the brain may be important physiologically. Our results suggest that in addition to cleaving biocytin, biotinidase can be biotinylated in the presence of biocytin at neutral and alkaline pH. Moreover, biotinidase may act as a biotinyl-transferase if biotinylated biotinidase is incubated with appropriate nucleophilic acceptors. Note added in proof Identical results were obtained using goat anti-biotin peroxidase conjugate (Sigma, St. Louis, MO) instead of avidin-peroxidase.

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References [1] Moss J, Lane MD. The biotin-dependent enzymes. Adv Enzymol 1971;35:321-442. [2] Wolf B, Grier RE, Secor McVoy Jr, Heard GS. Biotinidase deficiency: a novel vitamin recycling defect. J Inherit Metab Dis 1985;8(Suppl. 1):53-58. [3] Chauhan J, Dakshinamurti J. Purification and characterization of human serum biotinidase. J Biol Chem 1986;261:4268-4274. [4] Wolf B, Grier RE, Allen RJ, Goodman SI, Kien CL. Biotinidase deficiency: the enzymatic defect in late-onset multiple carboxylase deficiency. Clin Chim Acta 1983;131:273-281. [5] Baumgartner ER, Sourmala T, Wick H, Bonjour JP. Biotin-responsive multiple carboxylase deficiency (MCD): deficient biotinidase activity associated with renal loss of biotin. J Inherit Metab Dis 1984;7(Suppl. 2):123-125. [6] Wolf B, Miller JB, Hymes J, Secor McVoy J, lshikana Y, Shapira E. Immunological comparison of biotinidase in serum from normal and biotinidase-deficient individuals. Clin Chim Acta 1987;164:27-32. [7] Harlow E, Lane D. In: Antibodies: a laboratory manual. New York: Cold Spring Harbor Laboratory, 1988:298-299. [8] Hart PS, Hymes J, Wolf B. Biochemical and immunological characterization of serum biotinidase in profound biotinidase deficiency. Am J Hum Genet 1992;50:126-136. [9] Pispa J. Animal biotinidase. Ann Med Exp Biol Fenn 1965;43(Suppl. 5):1-39. [10] Chauhan J, Dakshinamurti K. The role of human serum biotinidase as biotin-binding protein. Biochem J 1988;256:265-270. [11] Mock DM, Lankford G. Studies of the reversible binding of biotin to human plasma. J Nutr 1990;120:375-381. [12] Cumming AM, Wesley RT. Analysis of von Willebrand factor multimers using a commercially available enhanced chemiluminescence kit. J Clin Pathol 1993;46:470-473. [13] Seehafer JG, Slupsky JR, Tang SC, Masellis-Smith A, Shaw ARE. Myristic acid is incorporated into the two acylatable domains of the functional glycoprotein CD9 in ester, but not in amide bonds. Biochim Biophys Acta 1990;1039:218-226. [14] Hayakawa K, Oizumi J. Human serum biotinidase is a thiol-type enzyme. J Biochem 1988;103:773-777. [151 McDonald A J, Mascagni F, Riley YD, Neal RL, Brinley-Reed M. Biocytin injections produce selective neuronal labeling in the rat CNS. Neuro Rep 1992;3:337-340. [16] Wolf B. Disorders of biotin metabolism: treatable neurological syndromes. In: Rosenberg R, Prusiner SB, Di Mauro S, Barchi RL, Kunkel LM, eds. The molecular and genetic basis of neurological disease. Stoneham, MA: Butterworth Publishers, 1992;569-581.