(Received for publication, August 19, 1994; and in revised form, October 25, 1994)
From the
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/-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.
Lysosomal protective protein was initially identified as a
glycoprotein regulating the expression of -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 ()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-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.
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.
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,
-galactosidase; d,
-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.
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 -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-terminal and
COOH-terminal oligopeptides of the human protective protein. (
)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. (
)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. 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. (
)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.