©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of the Disulfide Bond Involved in Post-translational Processing of Glycosylasparaginase and Disrupted by a Mutation in the Finnish-type Aspartylglycosaminuria (*)

(Received for publication, November 10, 1994)

Ashley L. McCormack Ilkka Mononen (1) Vesa Kaartinen (2) John R. Yates III (§)

From the  (1)Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195, the Department of Clinical Chemistry, Kuopio University Hospital, Fin-70211 Kuopio, Finland, and the (2)Department of Pathology and Laboratory Medicine, Children's Hospital of Los Angeles, Los Angeles, California 90054-0700

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The heavy chain of human glycosylasparaginase (N^4-(beta-N-acetylglucosaminyl)-L-asparaginase (EC 3.5.1.26)) has five cysteinyl residues (Cys-61, Cys-64, Cys-69, Cys-163, and Cys-179). A Cys-163 to serine substitution due to a point mutation in the glycosylasparaginase gene causes the most common disorder of glycoprotein degradation, the Finnish-type aspartylglycosaminuria. To localize the potential disulfide bonds, the isolated heavy chain of human leukocyte glycosylasparaginase was treated with the enzyme alpha-chymotrypsin, and the resulting peptides were separated by high performance liquid chromatography prior to and after reduction and S-carboxymethylation. The peptide containing the Cys-163 residue and the peptide to which it is connected with a disulfide were structurally characterized by mass spectrometry. The disulfide bond crucial for catalytic activity, subunit processing, and biological transport of glycosylasparaginase was located close to the carboxyl terminus of the heavy chain at positions 163 and 179.


INTRODUCTION

Glycosylasparaginase (glycoasparaginase, N^4-(beta-N-acetylglucosaminyl)-L-asparaginase (EC 3.5.1.26)) is a lysosomal enzyme that cleaves the linkage between N-acetylglucosamine and L-asparagine causing degradation of N-glycosidic glycopeptides. The structure, enzymatic characteristics, mechanism of action, and substrate specificity of human leukocyte glycosylasparaginase have been recently described(1, 2) . Glycosylasparaginase is encoded as an enzymatically inactive precursor polypeptide that is post-translationally processed to two subunits, 25-kDa heavy and 19-kDa light chains, which are connected by noncovalent forces(3) . The heavy chain has 5 cysteinyl residues (Cys-61, Cys-64, Cys-69, Cys-163, and Cys-179), and the light chain has 4 (Cys-286, Cys-306, Cys-317, and Cys-345), respectively. Two single-base changes in both alleles of the glycosylasparaginase gene, resulting in the replacement of arginine by glutamine (Arg-161 Gln) and cysteine by serine (Cys-163 Ser) on the heavy chain of the enzyme are found in 95% of the Finnish AGU (^1)patients(4, 5, 6) . The Cys-163 Ser substitution is known to destroy the catalytic activity, subunit processing, and lysosomal targeting of glycosylasparaginase (5) . This cleavage is responsible for activation of the enzyme (7) by creating the amino-terminal residue of the light chain, Thr-206, which is involved in the mechanism of enzyme action, (1) and is highly conserved among species(8) .

Aspartylglycosaminuria (McKusick 20840) is an autosomal recessive lysosomal storage disease caused by a deficiency in glycosylasparaginase. It is characterized by serious psychomotor retardation and accumulation of large amounts of glycoasparagines in body fluids and tissues(8) . AGU is the most common disorder of glycoprotein degradation and its frequency in the Finnish population (1:4000) is as high as that of the most commonly inherited lysosomal storage diseases (Tay-Sachs disease and Gaucher disease type I) among Ashkenazi Jews. In this paper we describe the localization of the disulfide bond in glycosylasparaginase, which is disrupted by the Cys-163 Ser mutation causing the Finnish-type AGU.


MATERIALS AND METHODS

Glycosylasparaginase was isolated from human leukocytes and purified 4600-fold(1) . The enzyme had a specific activity of 2.2 units/mg of protein. The light (19 kDa) and heavy (25 kDa) subunits of the native 88-kDa enzyme were isolated by reversed-phase HPLC. Peptides were produced from the heavy chain by treatment of the protein overnight with alpha-chymotrypsin in 50 mM ammonium bicarbonate, pH 8.6, containing 2 M urea and 5 mM iodoacetic acid. An aliquot of the digested material was analyzed by C(18) reversed-phase HPLC using a fused silica micro-column (198-µm inner diameter) and electrospray ionization mass spectrometry (LC-MS). A second aliquot was removed from the digested material, reduced for 1 h with dithiothreitol, and treated with iodoacetic acid. This aliquot was then analyzed by LC-MS.

Electrospray ionization mass spectrometry and tandem mass spectrometry were performed as described previously(9, 10) . The electrospray ionization tandem mass spectrometer, TSQ-700, was obtained from Finnigan MAT (San Jose, CA) and equipped with an Analytica ESI source (Branford, CT). The ABI 140B dual syringe pump (Applied Biosystems, Foster City, CA) was used for HPLC, and a sample injector with a 5-µl loop was obtained from Rheodyne (Cotati, CA). Peptides were eluted with a linear gradient of 5-90% Solvent B (80:20 acetonitrile, 0.5% acetic acid) over 30 min. The flow rate from the pumps was 100 µl/min. The solvent stream was split, 50:1-100:1, precolumn, and the final flow rate was 1-2 µl/min. Electrospray conditions were as follows: sheath gas, nitrogen at 0.2 liters/min; drying gas, heated nitrogen (80-85 °C, 16 psi) 4 liters/min; needle distance to capillary, 2.5 cm; capillary voltage, -3500-4100 V.

A 1-µl aliquot of sample was injected to record the molecular weight of the peptides produced by alpha-chymotrypsin digestion. Spectra were acquired by scanning the first mass analyzer or the second mass analyzer at a rate of 500 u/s over the range 400-1500 m/z. Sequence analysis of peptides was performed during a second HPLC analysis using tandem mass spectrometry with an equivalent amount of material. The peptide of interest was isolated by selecting the precursor ion with a 3-4-u (full width half height) wide window in the first mass analyzer and passing the ions into a collision cell filled with argon to a pressure of 3-5 millitorr. Collision energies (laboratory frame) were on the order of 20-50 eV. The fragment ions produced in the collision cell were transmitted to the second mass analyzer, which was scanned at 500 u/s over a mass range of 50 u to the molecular weight of the precursor ion to record the fragment ions. The electron multiplier setting was 200-400 V higher than used to record the ions in main beam. The second mass analyzer was tuned to give peaks which were 1.5 u wide.


RESULTS AND DISCUSSION

The amino acid sequence of human glycosylasparaginase heavy chain and the presumed cleavage sites produced by using alpha-chymotrypsin are shown in Fig. 1. The nonreduced protein was treated with the enzyme alpha-chymotrypsin in the presence of 5 mM iodoacetic acid, and the resulting mixture of peptides was analyzed by LC-MS. The total ion chromatogram is shown in Fig. 2A. An aliquot of the digested protein was reduced with dithiothreitol and S-carboxymethylated with iodoacetic acid. This mixture of peptides was analyzed by LC-MS (Fig. 2B), and an increase in the number of peptides was observed. A comparison of the m/z values of peptides expected upon treatment of the protein with alpha-chymotrypsin and those observed in the reduced and S-carboxymethylated mixture of peptides produced an ion that corresponds to the amino acid sequence LARNCQPNYW ((M + 2H), m/z 662) containing the Cys-163 residue mutated in AGU. Tandem mass spectrometry analysis of this ion confirmed the sequence assignment (Fig. 3). Calculation of the masses for all possible disulfide configurations with this peptide, and examination of the m/z values for the peptides observed in the LC-MS analysis of the nonreduced peptides, identified only one possibility. An ion of m/z 851 ((M + 2H)) corresponds to a peptide containing the sequences LARNCQPNYW and CGPY. This ion was not observed to an appreciable extent in the peptide mixture produced upon reduction and S-carboxymethylation. Tandem mass spectrometry analysis produced sufficient sequence information to confirm the assignment of LARNCQPNYW and CGPY as disulfide bridged peptides (Fig. 4). Thus, Cys-163 and Cys-179 are determined to be covalently bonded, and this bond is disrupted by mutation of Cys-163 Ser in the Finnish-type AGU(4, 5) . The present finding demonstrates on the protein level the cause of glycosylasparaginase deficiency in over 95% of cases of the most common disorder of glycoprotein degradation, the Finnish-type aspartylglycosaminuria. The presence of another potential disulfide bond between Cys-61, Cys-64, or Cys-69 could not be resolved since the residues are located close to one another and an alpha-chymotryptic cleavage site doesn't exist between the residues (Fig. 1). No other commercially available enzymes were capable of cleaving the amino acid residues necessary to determine the presence of additional disulfide bonds.


Figure 1: Amino acid sequence data on human glycosylasparaginase and localization of the disulfide bond disrupted by a mutation in the Finnish-type aspartylglycosaminuria. The amino acid residues of the heavy chain of human glycosylasparaginase are numbered as in (4) . The 5 cysteinyl residues are indicated in circles, and the presumed alpha-chymotrypsin cleavage sites are indicated in squares.




Figure 2: HPLC chromatogram of the heavy chain after digestion with alpha-chymotrypsin prior (A) and after (B) reduction and S-carboxymethylation. A, the peak marked L1 was shown to represent the peptide containing the disulfide bond between Cys-163 and Cys-179 (m/z, 851). B, the appearance of the S-carboxymethylated peptide containing Cys-163 is marked by L2 (m/z 662).




Figure 3: Tandem mass spectrum of the peptide LARNCQPNYW. Collision induced dissociation mass spectrum recorded on the (M + 2H) ions at m/z 662. Fragments of type b and y, ions having the general formulas H(NHCHRCO) and H(2)(NHCHRCO)OH, respectively, are shown above and below the deduced amino acid sequence at the top of the figure. Ions observed in the spectrum are underlined. Leu and Ile were assigned by correspondence to the nucleotide derived sequence. The spectrum was acquired using a collision energy of approximately 22 eV and a gas pressure of 3.5 millitorr. The second mass analyzer was scanned at a rate of 500 u/s.




Figure 4: Tandem mass spectrum of the peptide disulfide bonded peptides LARNCQPNYW and CGPY. Collision-induced dissociation mass spectrum recorded on the (M + 2H) ions at m/z 851. Fragments of type b and y, ions having the general formulas H(NHCHRCO) and H(2)(NHCHRCO)OH, respectively, are shown above and below the deduced amino acid sequence at the top of the figure. Ions observed in the spectrum are underlined. Leu and Ile were assigned by correspondence to the nucleotide derived sequence. The spectrum was acquired using a collision energy of approximately 10 eV and a gas pressure of 3.5 millitorr. The second mass analyzer was scanned at a rate of 500 u/s.



The presence of a disulfide bond between Cys-163 and Cys-179 suggests that stabilization or correct folding of the carboxyl terminus of the heavy chain of glycosylasparaginase by a disulfide bridge 26 residues prior to the cleavage site and Thr-206 (1, 7) is required for activation of the enzyme. The Thr-206 residue at the cleavage site is preceded by 11 hydrophilic residues at the carboxyl terminus of the heavy chain. These residues have been proposed to make the region susceptible to proteolytic hydrolysis in the lysosomes(3) . The Thr-206 residue is located at the amino terminus of the newly formed light subunit in a protein sequence that is highly conserved among species (8) and that is located at or close to the active site of glycosylasparaginase(1) . Stabilization and/or proper folding of the protein chain by a disulfide bond preceding the processing site may be of more general importance in studies for the processing mechanism for this enzyme, which is currently poorly understood. Whether it involves an actual processing protease or an autocatalytic cleavage remains to be shown.


FOOTNOTES

*
This work was supported by grants from the Sigrid Juselius Foundation, from the Medical Research Council of the Academy of Finland (to I. M.), and from the National Science Foundation, Science and Technology Center Cooperative Agreement 8809710 (to J. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biotechnology, FJ-20, University of Washington, Seattle, WA 98195. Tel.: 206-685-7388; Fax: 206-685-7344; jyates{at}u.washington.edu.

(^1)
The abbreviations used are: AGU, aspartylglycosaminuria; HPLC, high performance liquid chromatography; LC-MS, electrospray ionization mass spectrometry; u, atomic mass units.


REFERENCES

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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.