©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protective Protein as an Endogenous Endothelin Degradation Enzyme in Human Tissues (*)

(Received for publication, August 19, 1994; and in revised form, October 25, 1994)

Kohji Itoh (§) Ryoichi Kase Michie Shimmoto Akira Satake Hitoshi Sakuraba Yoshiyuki Suzuki

From the Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

An enzyme hydrolyzing the carboxyl terminus of endothelin-1 was detected in control human tissues but was deficient in tissues from a patient with galactosialidosis, a metabolic disease caused by the protective protein gene mutation. It was proportional to the amount of immunologically estimated mature protective protein. An antibody against the lysosomal protective protein/beta-galactosidase complex precipitated the enzyme activity almost completely. Transfection of the human cDNA for protective protein resulted in high expression of the enzyme activity in transformed fibroblasts from a galactosialidosis patient. These results indicated that the mature protective protein is a major soluble endogenous endothelin degradation enzyme in human tissues.


INTRODUCTION

Lysosomal protective protein was initially identified as a glycoprotein regulating the expression of beta-galactosidase (EC 3.2.1.23) and neuraminidase (EC 3.2.1.18) through the formation of a high molecular weight multienzymic complex in lysosomes(1, 2, 3, 4) . It is synthesized in human fibroblasts as a single precursor and then processed to a mature form, consisting of 32- and 20-kDa peptides linked by disulfide bonds(1) . A genetic defect of the protein causes a human inherited metabolic disease (galactosialidosis) with a secondary deficiency of the two enzymes and results in heterogeneous phenotypic expression(1, 5, 6) . Most patients develop neurological abnormalities, dysmorphism, and angiokeratoma(5, 6) .

On the basis of its sequence homology to yeast carboxypeptidase Y and the KEX1 (^1)gene product(7, 8) , this protein has been shown to possess serine esterase activities: that of carboxypeptidase at acidic pH, and those of esterase and carboxyl-terminal deamidase at neutral pH(9, 10) . All these enzyme activities are deficient in galactosialidosis cells(11, 12) . However, their physiological significance is not known at present.

The endothelins are a group of peptides that exert potent vasoconstrictive activity. They cause common long lasting hemodynamic, cardiac, and renal effects in many species(13, 14, 15) . Endothelin-1, originally derived from aortic endothelial cell cultures(13) , is expressed not only in the vasculature but also widely in various non-cardiovascular tissues, including the central and peripheral nervous systems(16, 17, 18) . Its plasma level is elevated in pulmonary hypertension(19) , myocardial infarction(20) , and acute renal failure (21) .

Each of the three endothelin peptides is synthesized as a preprotype precursor and then processed to an intermediate precursor (big endothelin), and finally to an active mature form by a specific metalloendopeptidase(13, 22) . All mature peptides share unusual common structural characteristics. They consist of 21 amino acids, with two intramolecular disulfide bonds on the NH(2)-terminal side and a conserved hydrophobic sequence, Ile-Ile-Trp, at the COOH-terminal end(15) . Removal of Trp from the COOH terminus of endothelin-1 abolishes its vasoconstrictive activity(23, 24) .

Recently, Jackman et al.(25) purified a deamidase from human platelets that modified the carboxyl termini of some bioactive peptides like tachykinins, and demonstrated its sequence identity with lysosomal protective protein. They also demonstrated that it preferentially removed the carboxyl-terminal Trp residue of endothelin-1 at acidic pH in vitro(26) . In another study, Deng et al.(27) purified an endothelin degradation enzyme (EDE) from rat kidney that has the biochemical characteristics and amino acid composition similar to those of the protective protein.

In this study, we demonstrated that a soluble EDE is identical to the protective protein. The distribution of the EDE activity in human tissues was the same as that of the mature protective protein.


EXPERIMENTAL PROCEDURES

Materials

Endothelin-1 was purchased from Novabiochem (Läufelfingen, Switzerland), phenylmethylsulfonyl fluoride (PMSF), and N-benzyloxycarbonyl-L-phenylalanyl-L-leucine (Z-Phe-Leu) from Sigma (St. Louis, MO), TES from Merck (Darmstadt, Germany), and leupeptin from the Peptide Institute (Osaka, Japan).

Autopsy Tissues and Culture Cells

Autopsy tissues from a galactosialidosis patient (type II; (28) ) and a Gaucher disease patient (pathological control) had been stored at -80 °C until use. Fibroblasts from a galactosialidosis patient were transformed with simian virus-40-adenovirus recombinant (ASVGS-1) (29) and cultured in Ham's F-10 medium supplemented with 10% fetal calf serum and antibiotics.

Immunoprecipitation of EDE Activity with an Anti-Protective Protein Antibody

The tissues were homogenized with a microhomogenizer (Niti-on, Chiba, Japan) in four volumes of 20 mM sodium acetate buffer (pH 5.6), containing 0.1 M NaCl in the absence or presence of 5 mM EDTA or 1 mM leupeptin, and then centrifuged at 10,000 times g for 15 min at 4 °C. The supernatant (5 µl) was incubated with 60 µg of anti-human lysosomal protective protein/beta-galactosidase complex (anti-complex) IgG (4) in 20 mM sodium acetate buffer (pH 5.6), containing 0.1 M NaCl (final volume, 25 µl), and then left for 2 h at 4 °C. Preimmune IgG was used as a control. Then, 20 µl of 50% (v/v) protein A-Cellulofine (Seikagaku Kogyo, Tokyo, Japan) in the same buffer was added. After incubation for 1 h at 4 °C with shaking, the mixture was centrifuged at 10,000 times g for 5 min. The EDE activity remaining in the supernatant was measured as described below.

Enzyme Assays

The EDE activity was measured by high performance liquid chromatography (HPLC) with endothelin-1 as a substrate according to the method of Deng et al.(27) . Endothelin-1 (500 pmol) was incubated with an aliquot of a cell or tissue extract in 50 mM TES, pH 6.0, for 10 min at 37 °C in a total volume of 15 µl. The reaction was terminated by adding 95 µl of 28% (v/v) acetonitrile in 0.09% (v/v) trifluoroacetic acid. Degradation products in the reaction mixture were separated on a 3.9 times 150 mm Nova-Pack C(18) column (Waters, Milford, MA) in an HPLC apparatus (Shimadzu, Kyoto, Japan), using a 10-min gradient of 22-35% acetonitrile in 0.09% trifluoroacetic acid, followed by 20 min of isocratic elution with 35% acetonitrile in 0.09% trifluoroacetic acid, at a flow rate of 1.1 ml/min at 40 °C. The absorbance of peptides was monitored at 215 nm. EDE activity was calculated from the rate of disappearance of endothelin-1. The degradation product was estimated as des-Trp endothelin-1, based on the retention time relative to that of the authentic endothelin-1, according to a previous report(27) . Carboxypeptidase activity was measured at pH 5.6, using Z-Phe-Leu as a substrate(12) . beta-Galactosidase and beta-glucuronidase activities were measured at pH 4.5 with 4-methylumbelliferyl derivatives as substrates(30) . Neuraminidase activity was not measured in the present study because of its instability. Protein determination was performed with a DC assay kit (Bio-Rad), using bovine serum albumin as a standard.

Transient Expression of Protective Protein cDNA in Transformed Fibroblasts

ASVGS-1 cells on 15-cm dishes at subconfluence were transfected with human protective protein cDNA in the expression vector, pCAGGS (75 µg of plasmid DNA), by the calcium phosphate-mediated transfection method, as described previously(29) . Cell extracts were prepared by sonication, twice for 10 s each, in 20 mM sodium acetate buffer (pH 5.6), containing 0.1 M NaCl, 5 mM EDTA, and 1 mM leupeptin, followed by centrifugation at 10,000 times g for 15 min at 4 °C. Enzyme activities and protein concentrations were assayed in the supernatant. A control experiment was performed using untransfected cells. In some experiments, the extract was treated with PMSF, a carboxypeptidase inhibitor(9) , before the enzyme assays.


RESULTS

EDE Activity in Human Tissues

EDE activity was observed in kidney, lung, and liver from a pathological control patient (Table 1), but was low in brain (data not shown). It was deficient in liver from a galactosialidosis patient. The amount of enzyme activity was proportional to those of the acid carboxypeptidase and beta-galactosidase activities. beta-Glucuronidase activity was independent of the EDE activity.



Immunoprecipitation of EDE Activity

HPLC gave a new peak, probably of des-Trp endothelin-1(26, 27) , after incubation of endothelin-1 with the tissue extract (Fig. 1). Most of the EDE activity in soluble extracts of kidney, liver, and lung was immunoprecipitated by the anti-complex antibody, and the HPLC peak of the degradation product disappeared. The result was the same regardless of the addition of EDTA or leupeptin in the extract (data not shown).


Figure 1: Immunotitration of EDE activity. The IgG fraction prepared from anti-complex antiserum or preimmune serum (60 µg) was added to a human tissue extract prepared as described under ``Experimental Procedures.'' The immune complex was removed by the addition of protein A-Cellulofine, and then the EDE activity remaining in the supernatant was measured. Panel A, kidney; panel B, lung; panel C, liver. a, treatment with preimmune IgG; b, treatment with anti-complex IgG. Peak 1, endothelin-1; peak 2, its degradation product.



Expression of Protective Protein cDNA in Galactosialidosis Cells

The activity was totally deficient in transformed galactosialidosis fibroblasts (Fig. 2). It was highly expressed after transfection with human protective protein cDNA (29 nmol/min/mg protein at pH 6.0 and 37 °C). Acid carboxypeptidase and beta-galactosidase activities also were elevated to the same degree, but beta-glucuronidase activity showed no change. The hydrolysis was inhibited by PMSF (data not shown).


Figure 2: Restoration of EDE activity in a galactosialidosis cell line transfected with protective protein cDNA. Transformed fibroblasts derived from a galactosialidosis patient (29) at subconfluence on a 15-cm dish were transfected with human protective protein cDNA (75 µg of plasmid DNA) by the calcium phosphate method. After 60 h, the cells were harvested and frozen at -80 °C. A cell extract was prepared by thawing and sonication. Panel A, enzyme activities in the extract. a, EDE; b, carboxypeptidase; c, beta-galactosidase; d, beta-glucuronidase. +, transfected cell extract; -, untransfected cell extract. Each value is the mean of duplicate measurements. Panel B, HPLC pattern of endothelin-1 hydrolysis. a, Treatment with the untransfected galactosialidosis cell extract (2.3 µg of protein); b, treatment with the galactosialidosis cell extract transfected with protective protein cDNA (3.0 µg of protein). Peak 1, endothelin-1; peak 2, its degradation product.




DISCUSSION

The protective protein is a multifunctional glycoprotein with at least two independent biological functions: first, a protective effect as to regulation of the expression of beta-galactosidase and neuraminidase in lysosomes(1, 2, 3, 4) ; and second, catalytic activities as to hydrolysis of peptide or ester bonds, i.e. those of as acid carboxypeptidase (optimal pH 5.6), neutral esterase (optimal pH 7.0), and carboxyl-terminal deamidase (optimal pH 7.0)(9, 10) . It is homologous to serine carboxypeptidases derived from yeast (7, 8) and plants(31, 32) . The proteins exhibiting this enzyme activity have a common catalytic triad, consisting of Ser, His, and Asp residues(8) , and are strongly inhibited by serine protease inhibitors(9, 10) . The catalytic activities of the protective protein were separated from the protective activity(10) .

Galactosialidosis is an inherited metabolic disease caused by a genetic defect of the protective protein, resulting in a deficiency of all the hydrolase activities(1, 5, 6, 12) . Sialylated glycoconjugates accumulate in the tissues (5, 6) and urine of the patients(33, 34) . The clinical manifestations are heterogeneous. In most cases, loss of vision appears as an initial symptom in the teens, and then characteristic neurosomatic manifestations follow in subsequent years (type II): neurological abnormalities represented by action myoclonus and cerebellar ataxia, skeletal dysplasia, cherry-red spots, and angiokeratoma(5) . Clinically severe cases (type I) have also been reported, with edema, ascites, skeletal dysplasia, and severe developmental retardation occurring in early infancy(6) . Several gene mutations have been identified for each clinical subtype(29) , and a correlation has been shown between the genotype and phenotype(29) . However, the relation between individual manifestations and gene mutations is not clear. Furthermore, the pathogenetic significance of the deficient enzyme activities of the protective protein is not known at present.

Recently, we raised two polyclonal antibodies against synthetic NH(2)-terminal and COOH-terminal oligopeptides of the human protective protein. (^2)Considerable amounts of the mature protein were detected on immunoblotting in human kidney, lung, and liver, but only a little in brain. Further immunohistochemical analysis revealed cell type-specific enrichment of the protein. (^3)The distribution was similar to that of endothelin-1(16, 17, 18, 35, 36, 37) .

In the present study, we obtained direct evidence for hydrolysis of endothelin-1 by the mature protective protein. First, cells and tissues from galactosialidosis patients did not show EDE activity; second, expression of cDNA for the protective protein restored the EDE activity in enzyme-deficient cells; third, the EDE activity was well correlated with the distribution of the mature protective protein; fourth, most of the EDE activity was immunoprecipitated by the anti-complex antibody. The mature protective protein is probably the major soluble endogenous EDE in human tissues.

The synthetic pathway for active mature endothelins has been studied in detail(22) , but less is known about its degradation at present. A membrane-bound neutral endopeptidase has been shown to be associated with the degradation in vivo(38) . Endothelins are relatively resistant to non-selective degradation by peptidases because of their unique structures. Their plasma levels are elevated in various disease states(19, 20, 21) . They are inactivated on removal of their carboxyl-terminal residues(23, 24) . It is possible that the carboxypeptidase activity of the protective protein regulates the endothelin activity. If endothelins are natural substrates of the protective protein as a carboxypeptidase, some clinical signs and symptoms may be explained on the basis of their metabolic error in patients with galactosialidosis.

At present, the site of endothelin degradation in vivo is not known. A granular pattern of immunostaining has been observed in somatic cells, suggesting its lysosomal distribution.^3 However, extracellular and pericellular actions of the protective protein have been suggested in previous studies(25, 26) . Recently, we purified and directly identified a serine carboxypeptidase secreted by human platelets as the protective protein. (^4)Further investigations of the intracellular and extracellular distributions of the protective protein will reveal its functional significance as to metabolic regulation of endothelins under physiological and pathological conditions, including galactosialidosis.


FOOTNOTES

*
This research was supported by grants from the Ministry of Education, Science and Culture (Japan), the Ministry of Health and Welfare (Japan), the Yamanouchi Foundation for Research on Metabolic Disorders, the Naito Research Foundation, and the Uehara Memorial Foundation. 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 Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-3823-6008.

(^1)
The abbreviations used are: KEX, killer expression; EDE, endothelin degradation enzyme; PMSF, phenylmethylsulfonyl fluoride; Z-Phe-Leu, N-benzyloxycarbonyl-L-phenylalanyl-L-leucine; TES, N-tris(hydroxymethyl)-2-aminoethanesulfonic acid; anti-complex, anti-human lysosomal protective protein/beta-galactosidase complex; HPLC, high performance liquid chromatography.

(^2)
A. Satake, K. Itoh, M. Shimmoto, T. C. Saido, H. Sakuraba, and Y. Suzuki, unpublished data.

(^3)
O. Soma, O. Sohma, M. Miziguchi, S. Takashima, A. Satake, K. Itoh, H. Sakuraba, Y. Suzuki, and K. Oyanagi, unpublished data.

(^4)
K. Itoh, N. Yamamoto, K. Tanoue, N. Takiyama, A. Satake, H. Sakuraba, and Y. Suzuki, unpublished data.


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