Báo cáo Y học: Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin doc

Based on the observation that the developmental pattern of the particulate liver TRH-DE and the serum TRH-DE are almost identical it has been proposed that the serum TRH-DE, like most se

Purification and characterization of the thyrotropin-releasing

hormone (TRH)-degrading serum enzyme and its identification

as a product of liver origin

Stephanie Schmitmeier1,*, Hubert Thole1,†, Augustinus Bader2and Karl Bauer1

1

Max-Planck-Institut fu¨r experimentelle Endokrinologie, Hannover, Germany;2Gesellschaft fu¨r Biotechnologische Forschung, Abt Organ und Gewebekulturen, Braunschweig, Germany

Previous biochemical studies have indicated that the

mem-brane-bound thyrotropin-releasing hormone

(TRH)-degra-ding enzyme (TRH-DE) from brain and liver and the serum

TRH-DE are derived from the same gene These studies also

suggested that the serum enzyme is of liver origin The

present study was undertaken to verify these hypotheses In

different species, a close relationship between the activities of

the serum enzyme and the particulate liver enzyme was

noticed The activity of the serum enzyme decreased when

rats were treated with thioacetamide, a known hepatotoxin

With hepatocytes cultured in a sandwich configuration,

release of the TRH-DE into the culture medium could also

be demonstrated The trypsin-solubilized particulate liver

TRH-DE and the serum TRH-DE were purified to

elec-trophoretic homogeneity Both enzymes and the brain

TRH-DE were recognized by a monoclonal antibody

gen-erated with the purified brain enzyme as antigen Lectin blot

analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligo-mannose/hybrid glycostructure A molecular mass of

97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting Fragment analysis of the serum TRH-DE revealed that the peptide sequences correspond to the cDNA deduced amino-acid sequences of the membrane-bound brain TRH-DE, whereby two peptides were identified that are encoded by exon 1 These data strongly support the hypo-thesis that the TRH-DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme

Keywords: TRH-degrading enzyme (TRH-DE); serum; liver; brain; characterization

The signal substance thyrotropin-releasing hormone

(TRH), a hypothalamic hypophysiotropic neuropeptide

hormone (reviewed in [1,2]) and a peptidergic

neurotrans-mitter/neuromodulator (reviewed in [3,4]), is known to be

rapidly inactivated by the brain TRH-degrading enzyme

(TRH-DE), an ectoenzyme located on the surface of

neuronal cells, as well as by the soluble serum TRH-DE

The highest activity of the membrane-bound TRH-DE is

found in brain and significant activities are also detected in

retina, lung and liver but not in other tissues such as heart,

kidney and muscle [5–7] Because the membrane-bound brain TRH-DE and the serum TRH-DE exhibit the same extraordinary high degree of substrate specificity and identical enzyme-chemical characteristics [8–14] it has been suggested that both enzymes are derived from the same gene

Based on the observation that the developmental pattern

of the particulate liver TRH-DE and the serum TRH-DE are almost identical it has been proposed that the serum TRH-DE, like most serum enzymes and proteins, might be

of liver origin [9,15] This interpretation was supported by the findings that the activities of the particulate liver enzyme, like the serum enzyme [16–18], is also regulated

by thyroid hormones [19] Moreover, similar enzyme-chemical properties between the particulate liver enzyme and the serum enzyme were noticed [9,15]

To verify the hypothesis that the serum TRH-DE is of liver origin we analyzed the TRH-DE in serum and tissue homogenates of different species and studied the effect of thioacetamide, a hepatotoxin, on the expression

of the serum enzyme and the particulate liver enzyme Furthermore, with hepatocytes in culture we analyzed the release of the TRH-DE into the medium Finally, we purified the TRH-DE from pig serum and liver to electrophoretic homogeneity and studied the relationship between these enzymes By sequence analysis we also verified the hypothesis that the membrane-bound brain TRH-DE and the serum TRH-DE are derived from the same gene

Correspondence to K Bauer, Max-Planck-Institut fu¨r experimentelle

Endokrinologie, PO Box 610309, D-30603 Hannover, Germany.

Fax: + 49 5115359 203, Tel.: + 49 5115359 200,

E-mail: karl.bauer@mpihan.mpg.de

Abbreviations: TRH, thyrotropin-releasing hormone; TRH-DE,

TRH-degrading enzyme; DFP, diisopropyl fluorophosphate; ECL,

enhanced chemiluminescence; SNA, Sambucus nigra agglutinin;

GNA, Galanthus nivalis agglutinin; MPSP, membrane

protein-solubilizing protease; TACE, TNFa protease.

*Present address: Department of Biochemistry and Molecular Biology,

University of Southern California, Keck School of Medicine and

Norris Comprehensive Cancer Center, Cancer Research Laboratory

#106, 1303 N Mission Road, Los Angeles, CA 90033, USA.

 Present address: Solvay Pharmaceuticals GmbH, PO Box 220,

D-30002 Hannover.

(Received 18 July 2001, revised 12 December 2001, accepted 8 January

2002)

M A T E R I A L S A N D M E T H O D S

Chemicals

Diisopropyl fluorophosphate (DFP), thioacetamide,

2-iodoacetamide, dithioerythritol and calf thymus DNA

were obtained from Sigma Aldrich Chemie GmbH

(Tauf-kirchen, Germany) Hoechst dye 33258 (bisbenzimidazol)

was from Calbiochem-Novabiochem GmbH (Bad Soden,

Germany) Glutamine and penicillin/streptomycin were

purchased from Life Technologies GmbH (Karlsruhe,

Germany) Insulin was from Hoechst AG (Frankfurt,

Germany), prednisone from MSD Sharp & Dohme GmbH

(Haar, Germany), and glucagon from Novo Nordisk

Pharma GmbH (Mainz, Germany) Poly(ethylene glycol)

6000 was obtained from Serva (Heidelberg, Germany)

Digoxigenin-labeled lectins, antidigoxigenin antibodies

con-jugated either to alkaline phosphatase or to horseradish

peroxidase as well as endoglycosidase F/N-glycosidase

F enzyme preparation and endoproteinase Lys-C were

purchased from Roche Diagnostics GmbH (Mannheim,

Germany) Goat anti-(mouse IgG) Ig conjugated to alkaline

phosphatase was obtained from Bio-Rad Laboratories

GmbH (Munich, Germany) The enhanced

chemilumines-cence (ECL)-Western blotting detection kit was from

Amersham Pharmacia Biotech (Freiburg, Germany)

Nitrocellulose BA-S83 was from Schleicher & Schuell

(Dassel, Germany) 5-Bromo-4-chloro-indolylphosphate

and Nitro blue tetrazolium were purchased from Biomol

Feinchemikalien GmbH (Hamburg, Germany)

Animals

Cows (Schwarz-bunte Rasse) and pigs (Deutsche

Land-rasse) were raised and maintained at the Institut fu¨r

Tierzucht und-verhalten, Mariensee, Germany Sprague–

Dawley rats were maintained at our institute according to

the animal welfare committee of the Medizinische

Hochsch-ule Hannover, Germany All animals had access to standard

chow and water ad libitum

Preparation of tissue homogenates and serum

After the animals were killed, blood and tissues were

immediately collected Serum was obtained after clotting

overnight at 4°C and centrifugation Livers were

thor-oughly perfused with cold NaCl/Pi (2.8 mM KH2PO4,

9.4 mM K2HPO4, 150 mM NaCl, pH 7.3) Brains and

perfused livers were minced and then homogenized in

3 vol of 10 mM sodium phosphate buffer, pH 7.3

containing 0.04% NaN3 (buffer A) by use of an Ultra

Turrax homogenizer (Jahnke and Kunkel, Staufen,

Ger-many)

Induction of liver cirrhosis in rats

Adult male Sprague–Dawley rats weighing 380–400 g

were used Over the experimental period 10 rats were

given tap water containing 0.03% thioacetamide and 10

rats were kept as control At given time intervals,

approximately 1 mL of blood was collected by

retrobul-bar puncture and after clotting serum was obtained by

centrifugation

Hepatocyte isolation and culture Hepatocytes were isolated from young male pigs (about 7 weeks old) as described previously [20] Isolated hepatocytes were adjusted to a density of 2· 106 viable cells per mL in Williams’ medium E supplemented with fetal bovine serum (10%), insulin (0.17 IUÆmL)1), predni-sone (0.85 lgÆmL)1), glucagon (0.015 lgÆmL)1), penicillin (100 UÆmL)1), streptomycin (100 lgÆmL)1) and glutamine (4.3 mM) The cells were plated onto 60-mm tissue culture dishes coated with collagen and then cultured as described

by Bader et al [21,22] The rate of albumin secretion into the culture medium was measured by electroimmu-nodiffusion [23] using a polyclonal antibody against porcine albumin Lactate dehydrogenase activity in the culture medium was determined by a modified method of Bergmeyer & Bernt [24]

Protein and DNA analysis The DNA content of cultured hepatocytes was determined according to the method described by Downs & Wilfinger [25] using the fluorescent dye bisbenzimidazol (Hoechst dye 33258) and calf thymus DNA as standard Protein was determined by a modification of the Lowry method as described by Peterson using bovine serum albumin as standard [26]

Determination of TRH-DE activity The assay was carried out as described previously using [pyroGlu-3H] TRH as substrate [27] In brief, samples were incubated at 30°C in a final reaction mixture of 50 lL containing 27 nM[pyroGlu-3H] TRH and the inhibitors of the cytosolic TRH-DEs (1 mMDFP and 1 mM 2-iodoacet-amide for post proline cleaving enzyme and pyroglutamate aminopeptidase, respectively) As a measure for the enzy-matic activity, the initial rate of TRH-degradation was determined by a four-point kinetic test

Purification of the TRH-DE from porcine serum Porcine serum (1 L) was diluted with 1 L of buffer A Under constant stirring, 2 L of a poly(ethylene glycol) 6000 solution (dissolved 50% w/v in buffer A) were added through a dropping funnel over a period of 3 h After an additional hour without stirring, the precipitated protein was pelleted by centrifugation at 17 000 g for 3 h The supernatant was discarded and the protein pellet was dissolved by stirring overnight with 3 L of buffer A The clear supernatant obtained after centrifugation at 17 000 g for 1 h was subjected to the purification procedure as described for the trypsin-solubilized membrane-bound TRH-DE from pig brain [28]

Purification of the membrane-bound TRH-DE from porcine liver

After homogenization of thoroughly perfused pig livers and washing of the membranes, the membrane-bound TRH-DE was solubilized by trypsin treatment and purified to homogeneity by following the protocol described for the isolation of TRH-DE from pig brain [28]

SDS/PAGE analysis

SDS/PAGE analysis was carried out according to Laemmli

[29] The proteins were denatured under reducing conditions

by boiling for 3 min in sample buffer containing 200 mM

dithioerythritol

Western blot analysis

After electrophoresis proteins were blotted onto a

nitrocel-lulose membrane as described by Towbin et al [30] After

blocking with NaCl/Tris (50 mMTris/HCl, 150 mMNaCl,

pH 7.5) containing 0.1% Tween-20 (NaCl/Tris/Tween), the

membrane was incubated overnight at 4°C with a

mono-clonal antibody (41H2; 4 lgÆmL)1) generated against the

particulate TRH-DE from pig brain The membrane was

then washed with NaCl/Tris/Tween and subsequently

incubated for 1 h at room temperature with goat

anti-(mouse IgG) Ig conjugated to alkaline phosphatase

(1 : 3000 in NaCl/Tris) After washing, the membrane was

incubated with a 5-bromo-4-chloro-indolylphosphate/Nitro

blue tetrazolium solution (335 lM

5-bromo-4-chloro-indo-lylphosphate, 400 lM Nitro blue tetrazolium in 200 mM

Tris/HCl, 100 mM NaCl, 10 mM MgCl2, pH 9.5) for

visualization

Lectin blot analysis

Lectin blot analysis was performed according to the method

described by Haselbeck et al [31] After Western blotting

and blocking, the membrane was cut and individual strips

were incubated for 1 h with digoxigenin-conjugated lectins

(1 : 1000 in NaCl/Tris containing 1 mM MgCl2, 1 mM

MnCl2, 1 mMCaCl2and 1 mMZnCl2, pH 7.5) The strips

were then washed with NaCl/Tris/Tween and subsequently

incubated for 1 h with sheep anti-digoxigenin Ig conjugated

either to alkaline phosphatase (0.75 UÆmL)1) or to

horse-radish peroxidase (0.1 UÆmL)1) After washing of the strips

with NaCl/Tris/Tween, the reaction products of alkaline

phosphatase or peroxidase were visualized by incubation

with the 5-bromo-4-chloro-indolylphosphate/Nitro blue

tetrazolium solution or by using the ECL-Western blotting

detection kit, respectively

Deglycosylation

Deglycosylation of the purified TRH-DE from liver, serum

and brain was performed as described previously [28] using

the endoglycosidase F/n-glycosidase F enzyme preparation

Briefly, the enzymes (30 lL containing 0.4–0.5 lg protein)

were denatured by boiling for 3 min in the presence of 0.1%

SDS After adding n-octylglycoside in a threefold excess to

SDS, the glycosidase mixture (0.2 U in 50 lL) was added

and the reaction mixture was incubated for 24 h at 25°C

Following SDS/PAGE and Western blotting, the enzymes

were visualized by using the monoclonal antibody 41H2 as

described above

Enzyme fragmentation and peptide sequencing

After isolation, the serum TRH-DE (100 lg, approximately

860 pmol) was either exposed to cyanogen bromide in 70%

formic acid or digested by endoproteinase Lys-C as

described for the particulate TRH-DE from rat and pig brain [32] Enzyme fragments were isolated by reverse-phase HPLC on C4 or C8 Vydac columns using acetonitrile in 0.1% trifluoroacetic acid as eluant Isolated fragments were analyzed by gas-phase sequencing using the Applied Biosystem 477A sequenator

R E S U L T S

Degradation of TRH by serum and tissue homogenates from different species

For comparative studies the specific activities of the TRH-DEs were determined in serum as well as in brain and liver homogenates from cow, pig and rat (Table 1) For all three species, high enzymatic activities were found in brain In rat and pig, high enzymatic activities were also detected in serum and significant activities in liver In contrast, very low activities were measured in liver homogenates and serum from cows

Table 1 Specific activities of the TRH-DEs in serum, liver and brain from rat, pig and cow Serum and tissue homogenates were prepared and analyzed as described in Materials and methods (n ¼ 3 for pig and cow, n ¼ 8 for rats, values are means ± SD).

Specific activity of the TRH-degrading enzyme (% TRH-degradedÆmin)1Æmg protein)1)

Rat 3.92 ± 0.45 0.95 ± 0.06 8.56 ± 1.05 Pig 11.92 ± 1.67 1.78 ± 0.26 10.27 ± 0.52 Cow 0.19 ± 0.03 0.04 ± 0.004 6.60 ± 0.51

Fig 1 Effect of thioacetamide, a hepatotoxin, on the activity of the serum TRH-DE Thioacetamide (0.03%) was either added or not to the drinking water of adult male rats At the indicated time points

1 mL of blood was collected from control (d) and thioacetamide-treated (s) animals Serum was prepared and tested for the TRH-degrading activity as described in Materials and methods (values are means±SD).

Influence of thioacetamide, a hepatotoxin,

on the activity of the serum TRH-DE

When rats were treated with thioacetamide, an agent which

is known to induce liver cirrhosis [33,34], we observed a

rapid decrease in the activity of the serum TRH-DE within

18 days (Fig 1) Histological examinations revealed

dis-tinctive evidence of damage in liver sections of animals

treated for 46 days In contrast to control animals, in liver

of thioacetamide-treated rats loss of the liver cell structure

and intercellular granula, increase of connective tissue and

appearance of noduli and fibrotic septa were noticed (data

not shown)

Synthesis of the TRH-DE by hepatocytes in culture

In contrast to hepatocytes kept as monolayers in primary

cultures, hepatocytes cultured in a sandwich configuration

continue to synthesize and secrete serum enzymes and

proteins after an initial lag phase required for cell recovery

[35,36] This lag phase is characterized by a decline of the

lactate dehydrogenase activity released into the culture

medium due to cell leakage (Fig 2) After 2 days of

cultivation and restoration of the cell membrane integrity,

the concentration of albumin and the activity of the

TRH-DE in the culture medium increased in a correlative

manner (Fig 2)

Purification of the TRH-DEs from serum and liver

The membrane-bound liver TRH-DE was solubilized and

purified by following exactly the procedure elaborated for

the purification of the particulate TRH-DE from rat and

pig brain [28] For the purification of the serum

TRH-DE fractionation by poly(ethylene glycol) precipitation

was used as the first step not only to partially purify the

enzyme but also to reduce the ionic strength due to salt

At a poly(ethylene glycol) concentration of 25% the serum enzyme completely precipitated The enzyme was recovered almost completely (97%) from the protein pellet and could be applied directly to the Q-Sepharose column Elution from this column and further purifica-tion followed again the procedure described previously [28]

Characterization of the TRH-DEs from serum and liver Molecular mass estimation An approximate molecular mass of 260 000 Da has been determined before for the serum TRH-DE by gel filtration of porcine serum [9] By the same method, a molecular mass of  250 000 Da could be estimated for the trypsin-solubilized and purified membrane-bound liver TRH-DE (Fig 3) When the purified enzymes were subjected to SDS/PAGE under reducing conditions a molecular mass of  125 000 Da could be estimated for both enzymes, the serum

TRH-DE and the trypsin-solubilized membrane-bound liver TRH-DE (Fig 4A), indicating that both enzymes consist

of two identical subunits In contrast, but in agreement with our previous data [28] a molecular mass of

116 000 Da could be determined for the

(Fig 4A)

Fig 2 Activity of the TRH-DE in the medium of cultured hepatocytes.

As described in Materials and methods pig hepatocytes were isolated

and seeded onto a collagen layer After cultivation for 24 h, the cells

were covered by a second layer of collagen (arrow) After gelatinization

of the second layer for 4 h, culture medium was added The medium

was changed every 24 h and used to determine the concentration of

albumin (r), the activity of lactate dehydrogenase (d) and the activity

of the TRH-DE (s) as described in Materials and methods (n ¼ 10;

values are mean±SD).

Fig 3 Estimation of the trypsin-solubilized TRH-DE from pig liver by gel filtration on a TSK-G 3000 SW-column After partial purification, the trypsin-solubilized membrane-bound liver TRH-DE was subjected

to gel filtration on a calibrated TSK-G 3000 SW-column The protein elution profile was monitored by following the absorbance at 280 nm (Æ Æ Æ) The enzyme activity (s) was determined as described in Materials and methods For calibration, a mixture of standard proteins (d) of known molecular mass, namely: thyroglobulin (1; M r 669 000), ferritin (2; M r 450 000), catalase (3; M r 245 000) and ovalbumin (4; M r 45 000) was applied to the same column.

Western blot analysis To verify the hypothesis that the

brain TRH-DE, the serum TRH-DE and the liver TRH-DE

are derived from the same gene, the enzyme preparations

were subjected to Western blotting All three enzymes were

recognized by the monoclonal antibody 41H2 which was

generated by using purified TRH-DE from pig brain as

antigen (Fig 4B) This finding indicates that these enzymes

are immunologically very similar At this point, it is worth noting that this antibody is specific to the enzymes of porcine origin and does not react with the enzymes from rat

or mouse

Identification as glycoproteins As in the case of the brain TRH-DE [28], the TRH-DEs from liver and serum also bind strongly to the Lentil-lectin Sepharose columns which were used for the purification of these enzymes Thus, both enzymes could be identified as glycoproteins

To gain more information as to the carbohydrate structure, the three enzymes were subjected to lectin blot analysis As shown in Table 2, the serum enzyme and the liver enzyme exhibit identical properties, distinctly differ-ent from the brain enzyme For example, the liver enzyme and the serum enzyme are readily recognized by the lectin SNA (Sambucus nigra agglutinin) but not by the lectin GNA (Galanthus nivalis agglutinin), whereas the opposite is true for the brain enzyme The collected data shown in Table 2 indicate that the brain enzyme con-tains an oligomannose/hybrid glycostructure, whereas the serum enzyme and the liver enzyme belong to the groups

of glycoproteins with a glycostructure of the complex type

To substantiate the notion that the TRH-DEs from liver, serum and brain differ only in the carbohydrate moiety, the three enzymes were incubated with the endoglycosidase F/N-glycosidase F enzyme preparation After Western blotting, a molecular mass of 97 000 Da could be determined for all three enzymes (Fig 4C) and thus a carbohydrate content of 22% could be estimated for the liver enzyme and the serum enzyme vs 16% for the brain enzyme

Peptide sequences of the serum TRH-DE

No sequence information could be obtained when the purified serum enzyme was subjected directly to sequencing, indicating that the aminoterminus is blocked Therefore, serum TRH-DE was either subjected to cyanogen bromide cleavage or to enzymatic digestion with endoproteinase Lys-C Overall 25 peptides could be isolated and sequenced Ten peptides are listed in Table 3 Interestingly, four peptides (3, 4, 8 and 10) were identical with the sequences determined before when fragments of the membrane-bound

Table 2 Lectin blot analysis of the TRH-DEs from pig brain, serum and liver The enzyme preparations were subjected to SDS/PAGE followed by Western blotting The nitrocellulose membrane was then cut and individual strips were incubated with digoxigenin-conjugated lectins Anti-digoxigenin antibodies conjugated either to alkaline phosphatase or to horseradish peroxidase were used for visualization as described in Materials and methods.

Lectin

TRH-degrading

Fig 4 SDS/PAGE and Western blot analysis of the purified porcine

TRH-DE from brain, liver and serum As described in Materials and

methods the solubilized and purified membrane-bound TRH-DE from

brain (lane 1) and liver (lane 3) as well as the purified serum TRH-DE

(lane 2) were subjected to SDS/PAGE and visualized either by silver

staining (A) or immunologically after Western blotting onto a

nitro-cellulose membrane by use of the monoclonal antibody 41H2 (B) For

the identification as glycoprotein (C), the purified enzymes were either

treated (T) or not (NT) with the endoglycosidase F/N-glycosidase F

enzyme preparation as described in Materials and methods and then

subjected to SDS/PAGE followed by Western blotting The proteins

were again identified by use of the monoclonal antibody 41H2.

TRH-DE from pig brain were analyzed [32] This result

clearly demonstrates that both enzymes are derived from the

same gene Comparison of the peptide sequences of the

serum TRH-DE with the cDNA deduced amino-acid

sequences of the TRH-DE from rat [32] and human [37]

brain reveals that only eight amino acids (2.8%) were

different out of the 288 amino acids identified, whereby at

six positions the amino acids of the enzyme from pig and

human were identical and different from the rat enzyme and

only at two positions were the amino acids of the porcine

peptide sequence different from that of rat and human,

which in turn were identical

D I S C U S S I O N

Even before TRH was finally isolated and structurally

elucidated, rapid inactivation of the biologically active

material by serum enzyme(s) had been demonstrated [38]

Subsequently, the serum enzyme catalyzing the hydrolysis

of TRH at the pyroGlu-His bond has been characterized

[8,9] The findings that the activity of this enzyme is

regulated by thyroid hormones [16–18] strongly suggest a

physiological role of this peptidase for the inactivation of

TRH released into the peripheral circulation This

inter-pretation is also supported by the high substrate specificity

of the enzyme [10] which therefore has also been named

ÔthyroliberinaseÕ The physiological importance of this

peptidase was also supported by the observation that the

TRH-degrading enzyme (TRH-DE) is absent in the plasma

of neonatal rats, whereas TRH is rapidly inactivated by

plasma of adult rats [39] The endocrinological importance

of this enzyme was subsequently questioned by the findings

that the activity of this peptidase varies considerably among

species and is almost absent in the plasma of beagle dogs [5]

In this study, the half-life of TRH after incubation with

various homogenates from different species was also

examined but a correlation between the half-life of TRH

and TRH-degrading activities of the tissue homogenates

between species was not observed This result is not

surprising as in tissue homogenates TRH is not only

inactivated by one enzyme as in serum or plasma but is

degraded by three peptidases (reviewed in [13,40]) namely

pyroglutamate aminopeptidase and post proline cleaving

enzyme (both are cytosolic enzymes), and the

membrane-bound TRH-DE, whereby the latter peptidase exhibits identical enzyme-chemical characteristics as the serum TRH-DE Using enzyme-specific conditions to determine the activity of the TRH-specific TRH-DEs indeed we found high enzymatic activities in brain homogenates of all three species examined In contrast, considerable differences in the TRH-degrading activities were noticed in the serum of these animals, whereby the enzyme activity is almost absent

in the serum from cow Interestingly, we also observed a correlation between the activity of the serum TRH-DE and the activity of the TRH-DE in liver homogenates, suggest-ing that the serum enzyme may be of liver origin

At present we do not have an explanation for the late development of the serum TRH-DE or for the extremely low activity of this enzyme in some species Nevertheless, these results support the notion that the serum TRH-DE, like most serum enzymes and proteins, is derived from the liver The decrease of the activity of the TRH-DE in rats treated with thioacetamide, a known hepatotoxin which induces liver cirrhosis [33,34] seemed to be in line with this interpretation However, a rapid decrease in the enzymatic activity was already observed within a few days after thioacetamide treatment As liver cirrhosis is generally a long-term process and is observed histolog-ically only after treatment with thioacetamide for several weeks, this decrease in the enzymatic activity seems to be related to other effects of thioacetamide on hepatocytes such as the reported inhibition of respiratory metabolism, binding to metal-containing enzymes, blockade of mRNA transport and loss of the cell’s ability to store glycogen [41]

Our experiments with hepatocytes in primary culture provided more direct evidence for the notion that the serum TRH-DE is of liver origin While hepatocytes in monolayer cultures appear to dedifferentiate and rapidly stop secreting liver-derived proteins, these cells maintain their function (e.g secretion of albumin, transferrin, a1-antitrypsin) when cultured in a collageneous matrix [20–22,35,36] After seeding and establishing the cultures in a sandwich confi-guration, we observed a decrease in the activity of lactate dehydrogenase (a marker for the restoration of the integrity

of the cell membrane) released into the culture medium Correspondingly, we found an increase in the amount of albumin (a liver specific marker protein) and an increase in

Table 3 Sequence of peptide fragments The serum TRH-DE was either subjected to cyanogen bromide cleavage (+) or digested with endopro-teinase Lys-C (à) The peptides were isolated by reverse-phase HPLC and sequenced The peptides which had been identified before from digests of TRH-DE from pig brain [32] are marked with an asterisk The numbers refer to the position of the amino acid as deduced from the cDNA of rat [32]

or human [37] brain TRH-DE Differences are found at position 604 (F in pig; L in human and rat), 607 (T in pig and human; M in rat), 614 (I in pig and human; L in rat), 680 (L in pig and human; I in rat), 760 (K in pig; R in rat and human), 991 (A in pig and human; S in rat), 1009 (M in pig and human; R in rat) and 1023 (L in pig and human; M in rat).

the activity of the TRH-DE released into the culture

medium, suggesting that the increase in the enzymatic

activity is due to the increased synthetic activity of

hepatocytes and not due to cell leakage

For direct analysis we purified the membrane-bound liver

TRH-DE after solubilization by trypsin and the serum

TRH-DE to electrophoretic homogeneity by following the

procedure described for the isolation of the

membrane-bound TRH-DE from pig brain [28] By gel filtration a

molecular mass of 250 000 Da could be estimated for the

truncated liver enzyme, a value which corresponds well with

the molecular mass of the papain-solubilized liver enzyme

[15] and the molecular mass of the serum enzyme [9]

reported before (260 000 Da) but differs from the molecular

mass of 230 000 Da determined for the trypsin-solubilized

brain enzyme [28] After SDS/PAGE under reducing

conditions a molecular mass of 125 000 Da was estimated

for the liver enzyme and the serum enzyme and a molecular

mass of 116 000 Da for the brain enzyme, indicating that all

these enzymes exist as homodimers, like many surface

peptidases [42] Interestingly, TRH-DEs from brain, liver

and serum were all recognized by the monoclonal antibody

41H2 which was generated after immunizing mice with the

TRH-DE from pig brain

As the brain TRH-DE has been identified as a

glycopro-tein [28], the immunological identity of this enzyme, the

serum enzyme and the liver enzyme strongly suggested that

these proteins differ only in their carbohydrate moiety

Analysis of the carbohydrate structures revealed that the

brain enzyme contains a glycostructure of the

oligoman-nose/hybrid type The occurrence of terminal

nonsubstitu-ted mannose and galactose residues is a general feature of

most brain glycoproteins [43] and thus the brain TRH-DE is

a ÔbrainÕ type glycoprotein [44] In contrast, TRH-DE from

both liver and serum contained terminal nonsubstituted

a(2–6)-sialic acid units linked to galactose and were thus

characterized as glycoproteins of the ÔserumÕ type [45] The

presence of sialic acid units in serum proteins is of biological

importance, as on hepatocytes desialyated proteins are

recognized by asialoglycoprotein-specific receptors and are

thus removed from the circulation by the liver [46] As

glycosylation is a species- and tissue-specific process [47–50]

the three enzyme preparations were enzymatically

degly-cosylated and subsequently subjected to SDS/PAGE For

all three enzymes a band with a molecular mass of

97 000 Da could be visualized immunologically, indicating

the polypeptide chain of these enzymes is very similar or

identical These results strongly support the hypothesis that

the serum TRH-DE and the membrane-bound TRH-DE

from brain and liver are derived from the same gene,

whereby the soluble enzyme might be either generated by

alternative splicing of the mRNA (e.g as reported for

immunoglobulin l [51,52]) or by proteolytic cleavage of the

membrane-bound liver enzyme as demonstrated for various

membrane-bound proteins with soluble counterparts

(reviewed in [53]) By fragmentation analysis of the purified

serum TRH-DE, two peptide sequences (peptide 1 and 2)

(Table 1) could be identified which correspond to the

sequences 160–173 and 192–221 of the cDNA deduced

amino-acid sequence of the membrane-bound brain

TRH-DE As both peptides are encoded by exon 1 which

ends at the position of amino-acid 260 [37,54], we can

conclude that the serum enzyme is not a product of

alternative mRNA splicing but must be generated by proteolysis Whether the serum enzyme is released from the plasma membrane of hepatocytes by proteases acting as sheddases or secretases (also designated as membrane protein-solubilizing proteases, MPSPs) [53,55–57] remains

to be elucidated Preliminary experiments indicate that the release of the serum enzyme is not affected by inhibitors directed against well characterized sheddases [namely b-secretase, c-secretase and TNFa protease (TACE)] The present results indicate furthermore that the serum enyzme might be generated intracellularly because after homogeni-zation of isolated hepatocytes and high speed centrifuga-tion, 40% of the TRH-degrading activity could be found in the cytosolic fraction and 60% of the enzyme activity was recovered from the particulate fraction (Schmitmeier, S & Bauer, K., unpublished observation) This aspect warrants further investigation

A C K N O W L E D G E M E N T S

We would like to thank Prof Dr P W Jungblut for his interest and encouragement and for providing the antibodies against serum albumin We also thank H O Bader, S Thiele for animal care,

P Affeldt for advice and help, and V Ashe for typing and for linguistic help in preparing the manuscript Supported by the Deutsche Forschungsgemeinschaft.

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