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Endocrinology Vol. 146, No. 9 3791-3799
Copyright © 2005 by The Endocrine Society

Expression of Neuroserpin Is Linked to Neuroendocrine Cell Activation

Dorien M. de Groot and Gerard J. M. Martens

Department of Molecular Animal Physiology, Institute for Neuroscience, Nijmegen Center for Molecular Life Sciences, Radboud University, 6525 GA Nijmegen, The Netherlands

Address all correspondence and requests for reprints to: Dr. G. J. M. Martens, Department of Molecular Animal Physiology, Institute for Neuroscience, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands. E-mail: g.martens{at}ncmls.ru.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitors of serine proteases (serpins) are important regulators of intracellular and extracellular proteolytic pathways, and they function by forming an irreversible complex with their substrate. Neuroserpin represents a neuroendocrine-specific serpin family member that is expressed in brain regions displaying synaptic plasticity. In this study, we explored the biosynthesis of endogenous neuroserpin in a neuroendocrine model system, namely the melanotrope cells of Xenopus intermediate pituitary. The biosynthetic activity of these cells can be physiologically manipulated (high and low production of the prohormone proopiomelanocortin in black and white animals, respectively), resulting from a synaptic plasticity in innervating hypothalamic neurons. We found that neuroserpin was also differentially expressed in the Xenopus intermediate, but not anterior, pituitary with a 3-fold higher mRNA and more than 30-fold higher protein expression in the active vs. the inactive melanotrope cells. Two newly synthesized glycosylated forms of the neuroserpin protein (47 and 50 kDa) were produced and secreted by the active cells. Intriguingly, neuroserpin was found in an approximately 130-kDa sodium dodecyl sulfate-stable complex in the active, but not in the inactive, melanotrope cells, which correlated with the high and low proopiomelanocortin expression levels, respectively. In conclusion, we report on the biosynthesis of neuroserpin in a physiological context, and we find that the induction of neuroserpin expression and the formation of the 130-kDa neuroserpin-containing complex are linked to neuroendocrine cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SERINE PROTEASES, such as plasminogen activators, plasmin, and thrombin, are known primarily for their role in blood coagulation and fibrinolysis. In addition, the proteolytic activity of these proteases is important for maintaining the integrity of the extracellular matrix (ECM). In the nervous system, serine proteases regulate plasticity in the developing as well as the mature brain (reviewed in Refs.1 and 2). For example, tissue-type plasminogen activator (t-PA) is expressed in the nervous system and has been implicated as playing a role in synaptic plasticity and memory development (3, 4, 5, 6, 7). Serine protease inhibitors (serpins) form a large family that can be found both intracellularly and extracellularly and regulates a number of physiological processes, such as coagulation, fibrinolysis, complement activation, angiogenesis, and apoptosis. The primary function of most members of this family is to neutralize intracellular and extracellular proteolytic pathways. One characteristic of the inhibitory serpin family members is the way in which they inhibit their substrate. Also known as suicide inhibitors, these serpins form sodium dodecyl sulfate-stable complexes with their target protease, and complex formation is considered to be biologically irreversible (reviewed in Ref.8). The rapid clearance of the serpin-protease complex takes place by binding of the complex to its receptor and subsequent internalization (9, 10).

Neuroserpin is a newly identified member of the serpin family primarily expressed in brain during late stages of development and during adulthood in regions that exhibit synaptic plasticity (11, 12, 13). Furthermore, neuroserpin has been identified in neuroendocrine cells, e.g. in rat pituitary and adrenal glands (14). t-PA has been a target protease for neuroserpin (13, 15, 16), although gene-targeting of neuroserpin in mice has implicated the inhibitor as playing a role in the regulation of emotional behavior through a mechanism that is, at least in part, independent of t-PA activity (17). Like other serpin family members, neuroserpin undergoes a marked conformational transition to be able to function as an inhibitor. Inevitably, this delicate process also renders the molecule susceptible to point mutations. Five mutations in the neuroserpin gene have been described, and they indeed lead to polymerization and accumulation of the protein, resulting in neuronal inclusion bodies and subsequent neuronal degeneration. This process underlies the recently described autosomal-dominant dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB) (Refs.18 and 19 and reviewed in Ref.20).

Thus far, the biosynthesis of only transfected recombinant neuroserpin has been studied (21). In this study, we examined the expression and biosynthesis of endogenous neuroserpin in the neuroendocrine melanotrope cells of the intermediate pituitary of Xenopus laevis. The process of background adaptation of this amphibian provides the opportunity to manipulate in a physiological way, in vivo, the activity of the melanotrope cells as well as their regulatory neuronal input. In animals adapted to a black background, the melanotrope cells are very active and produce vast amounts of the prohormone proopiomelanocortin (POMC), which is processed to a number of bioactive peptides, including {alpha}-MSH. This hormone causes pigment dispersion in skin melanophores, giving the animal a black appearance. In white-background-adapted animals, the activity of the melanotrope cells is inhibited by neurons, originating from the hypothalamic suprachiasmatic nucleus, which make direct synaptic contacts with the melanotrope cells. Thus, manipulation of melanotrope cell activity is accompanied by differences in synaptic plasticity (reviewed in Ref.22). Here we find that neuroserpin mRNA and protein expression are also induced in the biosynthetically active melanotrope cells from black-adapted animals compared with the inactive cells of white animals. Neuroserpin was synthesized as a 47-/50-kDa protein and formed, presumably with its substrate, a stable approximately 130-kDa complex, the formation of which was dependent on the color of the background of the animal and thus the melanotrope cell activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
South African claw-toed frogs, X. laevis, were reared in the Central Animal Facility of the Radboud University of Nijmegen (Nijmegen, The Netherlands). The animals were adapted to their background by keeping them in either white or black buckets under constant illumination for at least 3 wk at 22 C. All experiments were carried out in accordance with the European Communities Council Directive 86/609/EEC for animal welfare.

Antibodies
Four antineuroserpin antibodies were used: mouse monoclonal IgG and rabbit polyclonal IgG against human recombinant neuroserpin (m{alpha}-NS and {alpha}-NS-L, respectively; generous gifts of D. A. Lawrence, American Red Cross Holland Laboratory, Rockville, MD; Ref.23), rabbit polyclonal IgG against human recombinant neuroserpin ({alpha}-NS-M, generous gift of D. Lomas, University of Cambridge, Cambridge, UK; Ref.21), and rabbit polyclonal IgG directed against the C-terminal region of rat neuroserpin (CGRVMHPETMNTSGHDFEEL; {alpha}-NS-PEP) (generous gift of N. Birch, University of Auckland, New Zealand; Ref.14). The dilutions used were 1:400 ({alpha}-NS-L) and 1:300 (m{alpha}-NS and {alpha}-NS-PEP) for immunoprecipitation and 1:5000 ({alpha}-NS-L), 1:2500 (m{alpha}-NS), and 1:10000 ({alpha}-NS-M) for Western blotting. Tubulin, POMC, and p24{delta}2 were identified on Western blots using a monoclonal antitubulin antibody (1:1500) (generous gift of Dr. J. Fransen, Radboud University, Nijmegen, The Netherlands; Ref.24), a polyclonal anti-POMC antibody (1:20,000) (ST62; generous gift of Dr. S. Tanaka, Shizuoka University, Shizuoka, Japan; Ref.25), and the polyclonal anti-p24{delta}1/2 antibody 1262CH (1:5000) (as described previously in Ref.26), respectively.

Separation of Xenopus pars nervosa and intermediate pituitary
To compare neuroserpin protein expression in the pars nervosa with that in the intermediate pituitary, the two lobes were separated. To isolate the melanotrope cells from the neurointermediate lobe (NIL), NILs were dissected, washed several times in sterile Xenopus XL15 [10 mM glucose, 2 mM CaCl2, 1% kanamycin (Life Technologies, Inc.), 1% antibiotic/antimycotic (Life Technologies, Inc.) in 67% Leibovitz’s-15 medium (Life Technologies, Inc.)], and transferred to XL15 containing 0.25% (wt/vol) trypsin. After incubating for 45 min at 20 C, XL15 was added and the lobes were suspended by seven passages through a siliconized Pasteur’s pipet. The cell suspension was transferred to a syringe and filtered through a nylon filter (pore size, 60 µm). Finally, the melanotrope cells were collected by centrifugation (10 min at 6000 rpm), and the remaining pars nervosa was collected from the filter. Alternatively, the pars nervosa and intermediate pituitary were separated by dissection under a microscope. After separation, homogenates of cells and lobes were made in lysis buffer [250 mM sucrose, 1% Triton X-100, 10 mM Tris Cl, 1 mM EDTA (pH 7.4), 1 µM phenylmethylsulfonyl fluoride (PMSF), 0.1 mg/ml soybean trypsin inhibitor] using a glass homogenizer. After 20 min incubation on ice, the samples were centrifuged for 20 min (153,000 rpm, 4 C). Sample buffer [50 mM TrisCl (pH 6.8), 100 mM dithiothreitol (DTT), 2% SDS, 0.1% bromephenol blue, 10% glycerol] was added to the supernatant, and the samples were heated for 30 min at 37 C.

Western blotting
Homogenates of the NIL, the pars nervosa, intermediate pituitary, and isolated melanotrope cells were prepared as described above. Samples (corresponding to 25–33% of one NIL) were separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (0.45 µM Hybond-P; Amersham Pharmacia Biotech, Piscataway, NJ) using a Bio-Rad (Hercules, CA) mini-protean II cell system. Molecular weight markers were run alongside the samples to be analyzed. For immunodetection, blots were washed one time in PBS and incubated for at least 1 h in blocking buffer (5% skimmed milk and 1% Tween 20 in PBS). Subsequently, blots were incubated with primary antiserum in blocking buffer for 16 h (4 C). After three rinses in wash buffer (1% skimmed milk and 1% Tween 20 in PBS), blots were incubated in horseradish peroxidase-conjugated goat-antirabbit or mouse IgG (1:5000) for 1 h. After three rinses in wash buffer and one rinse in PBS, the proteins were visualized by incubating blots in Lumi-LightPLUS Western blotting substrate (Roche Diagnostics, Manheim, Germany) and by subsequent exposure to x-ray film (Eastman Kodak, Rochester, NY). For quantification of relative neuroserpin protein levels in black- compared with white-adapted animals, detection was performed using a BioChemi imaging system (UVP, Inc., Upland, CA), and signals were analyzed using LabWorks 4.0 software (UVP BioImaging Systems, UVP, Inc., Cambridge, UK). Total densities of 44-, 47-/50-, and 55-kDa neuroserpin products were normalized to the corresponding tubulin samples (n = 3). Subsequently, the values found for 44-kDa neuroserpin in white-adapted animals were set at 1, and relative protein levels of 44-, 47-/50-, and 55-kDa neuroserpin were calculated. The mean differences of 44- and 47-/50-kDa neuroserpin were plotted in a graph, and a paired t test was used for statistical analysis.

Metabolic cell labeling and immunoprecipitation
For metabolic cell labeling, NILs of black-adapted Xenopus were rapidly dissected and preincubated in Ringer’s medium [112 mM NaCl, 15 mM HEPES (pH 7.4), 2 mM KCl, 2 mM CaCl2, 2 mg/ml glucose, and 0.3 mg/ml BSA] for 15 min at 22 C. Radioactive labeling of newly synthesized proteins was performed by incubating the NILs in Ringer’s medium containing 5 mCi/ml Tran35S label (ICN Radiochemicals, Irvine, CA) for 30 or 90 min at 22 C. After the pulse labeling, NILs were rinsed in Ringer’s medium and, in case a chase was performed, incubated in Ringer’s medium supplemented with 0.5 mM L-methionine for 150 min at 22 C. Subsequently, lysates were prepared as described above. Immunoprecipitation was performed in TTD buffer (50 mM HEPES, 140 mM NaCl, 0.1% Triton X-100, 1% Tween 20, 1 mM EDTA, 1 mg/ml deoxycholate, 1 µM PMSF, 0.1 mg/ml soybean trypsin inhibitor) supplemented with SDS (final concentration of 0.075%) and antiserum. After overnight rotation at 4 C, immune complexes were precipitated with protein A-Sepharose (Amersham Pharmacia Biotech), washed four times with TTD buffer, and analyzed on a 10% SDS-polyacrylamide gel.

N-glycosidase F treatment
For protein deglycosylation, N-glycosidase F (which cleaves N-linked sugar chains from proteins) was used. The NIL lysates were boiled for 10 min in 6 mM HEPES (pH 7.4) containing 0.06% SDS and subsequently supplemented with 0.5% Nonidet P-40, 10 µg/ml soybean trypsin inhibitor, and 0.1 µM PMSF, and incubated with or without 40 mU/ml N-glycosidase F (Roche Diagnostics) for 1.5 h at 37 C.

Two-dimensional gel electrophoresis and protein analysis
For two-dimensional gel electrophoretic analysis, NILs were dissected and the tissues were homogenized using a glass potter in 40 mM Tris base (pH 9.5) and 10 mM Pefablock (Roche). Proteins were trichloroacetic acid precipitated, air-dried, and dissolved in lysis buffer [7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1% DTT, and 0.8% IPGphor buffer (pH 3–10; Amersham-Biosciences, Little Chalfont, Buckinghamshire, UK)]. The samples were separated by isoelectric point (pI) (pH 3–10 gradient in the first dimension) and subsequently by molecular weight on 10% SDS-PAGE. Neuroserpin products were then identified by Western blotting using the {alpha}-NS-L antibody. Protein spots were analyzed using matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF MS) and liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) as described previously (27).

Real-time quantitative RT-PCR analysis
The NIL and anterior lobe (AL) of the pituitary of black- and white-adapted Xenopus were dissected and collected in Trizol (Life Technologies), and total RNA was isolated according to the manufactures instructions. The RNA was then dissolved in 20 µl RNase-free H2O, and the concentration was measured with a GeneQuant RNA/DNA calculator (Biochem Ltd., Cambridge, UK). One microgram total RNA was reverse transcribed; first-strand cDNA synthesis was performed using 11 µl RNA and 1 µl pd(N)6 (random primers, 5 mU/µl; Roche) at 70 C for 10 min, followed by double-strand synthesis in 20 µl strand buffer (Life Technologies, Inc.) with 10 mM DTT, 20 U RNAsin (Promega, Madison, WI), 0.5 mM deoxyribonucleoside triphosphates (Roche), and 100 U reverse transcriptase (SuperScript II, Life Technologies) at 37 C for 90 min. Samples were diluted five times in RNase-free H2O and subjected to real-time quantitative RT-PCR on a 5700 GeneAmp PCR system (PE Applied Biosystems, Wellesley, MA) as follows: 5 µl template cDNA was added to 20 µl SYBR green buffer (PE Applied Biosystems) with 3 mM MgCl2, 0.2 m deoxyribonucleoside triphosphates (PE Applied Biosystems), 0.6 µM of each primer, and 0.625 U AmpliTaq gold (PE Applied Biosystems). To amplify the neuroserpin nucleotide sequence, the forward primer 5'-attgaaagcccatttgattgaag-3' and reverse primer 5'-tcaagacctcctttaggttcactactt-3' were used, and for glyceraldehyde-3-phosphate dehydrogenase, the primers 5'-gccgtgtatgtggtggaatct-3' and 5'-aagttgtcgttgatgacctttgc-3', respectively. PCR conditions were 95 C for 10 min, followed by 40 reaction cycles of 95 C for 15 sec, and 60 C for 1 min each. For each reaction, the cycle threshold (Ct) was determined, i.e. the cycle number at which fluorescence was detected above an arbitrary threshold (0.5). At this threshold, Ct values were within the exponential phase of the amplification. Ct values for neuroserpin mRNAs in lobes from black- vs. white-adapted animals were normalized to that of the internal standard (GADPH) mRNA. To examine the length of the amplified DNA, the reaction products were run on a 2.5% agarose gel and visualized with ethidium bromide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neuroserpin mRNA and protein expression in the pituitary of black- and white-adapted Xenopus
We used quantitative RT-PCR to examine neuroserpin mRNA expression in the NIL and in the AL of the pituitary of black- and white-adapted Xenopus. Neuroserpin mRNA was detected in both lobes of the pituitary. A 3-fold higher level of neuroserpin mRNA was found in the NIL of black-adapted animals than in that of white-adapted animals (3.2 ± 1.1-fold induction, n = 6). Neuroserpin mRNA levels were not significantly different in the ALs of black and white animals (Fig. 1Go). Thus, neuroserpin mRNA expression was up-regulated specifically in the biosynthetically active Xenopus melanotrope cells.



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  		FIG. 1. Neuroserpin mRNA levels in the neurointermediate and anterior pituitary of black- and white-adapted Xenopus. Neuroserpin mRNA levels in the NIL and the AL of the pituitary of black-adapted (BA; black bars) compared with those in white-adapted (WA; gray bars) animals were measured by quantitative RT-PCR. All values were normalized to GAPDH mRNA levels and are expressed as means ± SEM. A significant difference is indicated by an asterisk (P < 0.05).

 
Western blot analysis with a number of antineuroserpin antibodies ({alpha}-NS-L, {alpha}-NS-M, {alpha}-NS-PEP, and m{alpha}-NS) was performed to study the steady-state neuroserpin protein levels in the NILs of black- and white-adapted Xenopus. In NIL lysates of black-adapted animals, we detected with {alpha}-NS-L, {alpha}-NS-M, and m{alpha}-NS expression of approximately 44-, approximately 47-/50-, and approximately 55-kDa neuroserpin proteins. Furthermore, using the two polyclonal antineuroserpin antisera, {alpha}-NS-L and {alpha}-NS-M, an immunoreactive product of approximately 130 kDa was found (Fig 2AGo); the three additional products of approximately 57, approximately 60, and approximately 70 kDa are considered to be nonspecifically interacting products, as in each case they were recognized by only one of the two antibodies (Fig 2AGo, asterisks). The NS-PEP antibody was not appropriate for Western blot analysis, and the fact that the 130-kDa product was not detected with the monoclonal antibody m{alpha}-NS was due presumably to epitope masking. Using {alpha}-NS-L, {alpha}-NS-M, and m{alpha}-NS for analysis of NILs of white-adapted animals, only the 44-, 47-/50-, and 55-kDa proteins, but not the 130-kDa product, were found (Fig. 2AGo). At least a 30-fold higher level of expression of the 130-kDa neuroserpin-containing product was detected in the active melanotrope cells of black-adapted animals, compared with the inactive cells of white animals. The 44-kDa neuroserpin protein was up-regulated 3-fold in the NIL of black-adapted compared with white-adapted animals (2.8 ± 0.3-fold induction, n = 3), whereas 47-/50- and 55-kDa neuroserpin expression did not differ significantly (Fig. 2BGo and data not shown).



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  		FIG. 2. Neuroserpin protein levels in the neurointermediate pituitary of black- and white-adapted Xenopus. A, Lysates of neurointermediate lobes (NILs) of black- and white-adapted frogs (BA and WA, respectively) were separated by SDS-PAGE. After Western blotting, neuroserpin (NS) was identified using the polyclonal antineuroserpin antisera {alpha}-NS-L and {alpha}-NS-M and the monoclonal antineuroserpin antibody m{alpha}-NS. Tubulin was used as a control for loading equal amounts of protein. Nonspecifically bound proteins are indicated with asterisks. B, Quantification of 44- and 47-/50-kDa neuroserpin (NS) protein expression in NILs of black-adapted (BA) and white-adapted (WA) animals relative to 44-kDa neuroserpin expression in WA animals, which was set at 1. Values were normalized to tubulin protein levels and expressed as mean differences ± SEM. A significant difference is indicated by an asterisk (P < 0.05). C, The 130-kDa complex is expressed in the melanotrope cells of the intermediate pituitary of black-adapted Xenopus. Melanotrope cells of the intermediate pituitary were isolated from the NIL of BA and WA animals either by dissection of the pars intermedia (PI) tissue from the pars nervosa (PN) (dissected) or by trypsin treatment of the NIL tissue (enzymatic). Extracts of PI- and PN-tissues were separated on 10% SDS-PAGE and subjected to Western blot analysis with antineuroserpin antibody {alpha}-NS-L. Nonspecifically interacting proteins are indicated with asterisks. D, NIL lysate of black-adapted animals was treated with N-glycosidase F and subsequently subjected to SDS-PAGE and immunoblotting with {alpha}-NS-L.

 
The 130-kDa neuroserpin-containing complex is expressed in Xenopus intermediate pituitary cells
Because the NIL consists of the intermediate pituitary (the neuroendocrine melanotrope cells) and the pars nervosa (endings of neurons originating from the hypothalamus), we decided to study the site of neuroserpin protein expression in the Xenopus NIL in more detail. For this purpose, we separated the pars nervosa from the intermediate pituitary cells using two different strategies. First, the pars nervosa and intermediate pituitary were physically separated by dissection under a microscope. In a second approach, the melanotrope cells of the intermediate pituitary were isolated from the NIL by trypsin treatment; the remaining tissue after this treatment consisted mainly of the pars nervosa. Remarkably, we found that 55-kDa neuroserpin was expressed exclusively in the pars nervosa from black- and white-adapted Xenopus (Fig. 2CGo), whereas 47-/50-kDa neuroserpin and the 130-kDa complex were present in the melanotrope cells of the intermediate pituitary in black-adapted animals (Fig. 2CGo, upper panel). In the NIL of white-adapted animals, 47-/50-kDa neuroserpin expression levels were below the detection limit. The low amount of the 130-kDa product found in the pars nervosa after dissection or trypsin treatment probably originated from residual intermediate pituitary melanotrope cells, caused by incomplete separation of the two parts of the pituitary. The observed differences in the ratios between the 130-kDa complex and 55-kDa neuroserpin in the NIL (e.g. Fig. 2Go, A and D) were therefore probably caused by variations in tissue dissection (especially concerning the amount of pars nervosa tissue).

We next wanted to investigate whether neuroserpin is glycosylated in the Xenopus NIL. After N-glycosidase-F treatment of a NIL lysate, the migrations of the 130-kDa product and 47-/50-kDa neuroserpin shifted to products of approximately 115 and approximately 44 kDa, respectively, whereas the migration of 55-kDa neuroserpin was not affected (Fig. 2DGo). This finding indicates that only the 130-kDa product and 47-/50-kDa neuroserpin are N-linked glycosylated.

Biosynthesis and posttranslational modification of neuroserpin in the Xenopus neurointermediate pituitary
Pulse metabolic cell labeling of the Xenopus NIL for 30 or 90 min and subsequent immunoprecipitation analysis of the newly synthesized proteins with three antineuroserpin antisera ({alpha}-NS-L, NS-PEP,and m{alpha}-NS) revealed the production of two newly synthesized neuroserpin proteins with molecular masses of approximately 47 and approximately 50 kDa (Fig. 3AGo and data not shown). After a 2.5-h chase period, the newly synthesized neuroserpins were secreted into the incubation medium as products with slightly higher molecular masses (~48 and ~51 kDa) than the two neuroserpin products in the cells (Fig. 3BGo). Treatment of the immunoprecipitated newly synthesized neuroserpins with N-glycosidase-F gave an approximately 44-kDa product in both the cells and the medium, indicating that the 47- and 50-kDa neuroserpins and the two secreted forms are N-linked glycosylated (presumably on one residue in the 47-/48-kDa and two in the 50-/51-kDa forms) (Fig. 3CGo). The observed differences in molecular masses between the neuroserpins produced in the cells and secreted into the medium may thus be due to a modification of the N-linked sugar groups. After a 2.5-h chase period, but not after a 1-h pulse labeling, a product of approximately 130 kDa was immunoprecipitated from the cell lysates, suggesting that this product may represent a complex of newly synthesized neuroserpin with another protein and that this complex is not formed immediately after neuroserpin synthesis; we did not detect this complex in the media (Fig. 3CGo). Because treatment of the immunoprecipitate with N-glycosidase-F resulted in a shift of the newly synthesized 130-kDa to an approximately 115-kDa product, the complex was N-linked glycosylated (Fig. 3CGo), in line with the Western blot results (Fig. 2DGo).



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  		FIG. 3. Biosynthesis and release of neuroserpin by the intermediate pituitary cells of Xenopus. A, Neurointermediate lobes (NILs) were pulse labeled for 90 min. Cell lysates were directly analyzed on SDS-PAGE (total, representing 5% of the total cell lysate) or immunoprecipitated using the antineuroserpin antibodies {alpha}-NS-L, {alpha}-NS-PEP, or m{alpha}-NS, followed by resolving the immunoprecipitates with SDS-PAGE. When using m{alpha}-NS in the immunoprecipitation, a number of newly synthesized proteins, such as the proform of the prohormone convertase PC2 (proPC2) and POMC bound nonspecifically to the antibody (asterisks). B, NILs were pulse labeled for 90 min and chased for 2.5 h, and the cell lysate (c) and incubation medium (m) were directly analyzed on SDS-PAGE (totals, representing 5% of the total cell lysate and 20% of the incubation medium, respectively) or immunoprecipitated using {alpha}-NS-L, {alpha}-NS-PEP, or m{alpha}-NS, followed by SDS-PAGE. The secreted neuroserpin (NS) proteins and nonspecifically bound proteins are indicated with arrowheads and asterisks, respectively. C, NILs were pulse labeled for 60 min or pulse labeled for 60 min and chased for 2.5 h. Cell lysates and media were treated with N-glycosidase F before immunoprecipitation and subsequent SDS-PAGE. NS, Neuroserpin; proPC2, pro-prohormone convertase 2; POMC, proopiomelanocortin; deg., deglycosylated.

 
The 130-kDa neuroserpin-containing complex is acidic
Because of the striking characteristics of the 130-kDa neuroserpin-containing product (difference in expression levels in the NILs of black- and white-adapted Xenopus, and the newly synthesized 130-kDa product is not formed in the first hour of chase) we were interested in the nature of this high-molecular-mass product. In view of its size, it is not likely that the 130-kDa product would simply represent a multimerization of the 47-/50-kDa neuroserpin. One feature of serpins is their unusually strong binding to their substrate (reviewed in Ref.28). We hypothesized that the 130-kDa product might be an SDS-stable complex of neuroserpin with a serine protease such as t-PA, urokinase-type plasminogen activator (u-PA), plasmin, or thrombin, as these four proteins have been found to be in vitro substrates for neuroserpin (23, 13, 15). These potential substrates are basic proteins with a calculated pI of approximately 8.2, approximately 8.8, approximately 7.2, and approximately 7.3, respectively. To estimate the pI of the 130-kDa neuroserpin-containing complex in the NIL, two-dimensional gel electrophoresis of NIL protein extracts combined with immuodetection of neuroserpin was performed. We found that, in line with the calculated pI of Xenopus neuroserpin (pI 5.4), 47-/50-kDa neuroserpin, 55-kDa neuroserpin, and the 130-kDa complex are acidic products with a pI of approximately 5.6, approximately 5.2, and approximately 5.3, respectively (Fig. 4Go), indicating that the 130-kDa neuroserpin-containing complex displays a pI similar to that of neuroserpin itself. This observation implies that the binding partner of neuroserpin in the 130-kDa complex is also an acidic protein and makes it unlikely that, in the intermediate pituitary of black-adapted Xenopus, this complex is formed by binding of neuroserpin to the basic proteins t-PA, u-PA, plasmin, or thrombin.



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  		FIG. 4. Neuroserpin and the neuroserpin-containing complex are acidic in the neurointermediate pituitary of Xenopus. Two-dimensional gel electrophoresis (pH gradient in the first dimension and SDS-PAGE in the second dimension) of a lysate of the neurointermediate lobe of black-adapted Xenopus followed by Western blot analysis with antineuroserpin antibody {alpha}-NS-L.

 
In an attempt to identify the substrate of neuroserpin in the 130-kDa complex, we separated NIL proteins by two-dimensional gel electrophoresis, recovered a number of protein spots from the region in which the 130-kDa complex migrated, and analyzed the spots by MALDI-TOF-MS or by directly combined LC/ESI-MS. Unfortunately, the protein amounts were below the detection limit (data not shown), presumably because of the low expression level of the complex.

Dynamics in the expression of the 130-kDa complex in the intermediate pituitary during background adaptation of Xenopus
An interesting characteristic of X. laevis is its ability to adapt its skin color to its background. The process of background adaptation is mediated by {alpha}-MSH, a cleavage product of POMC that is produced at high levels in the intermediate pituitary melanotrope cells of a black-adapted animal. Because of the clear differential expression of the 130-kDa neuroserpin-containing complex in the inactive and active melanotrope cells of white and black animals, respectively, we were interested in the time course of the formation of this complex during background adaptation. For this analysis, the expression of the 130-kDa complex in the NIL was examined in white animals adapting to a black background and vice versa for various time periods. In white animals adapting to a black background, we first detected the complex after 5 d of adaptation; its level gradually increased until maximum expression levels were reached after 21 d, similar to the level found in fully black-adapted animals (i.e. adapted to a black background for >3 wk). The time course of expression of the 130-kDa complex was similar to that of POMC and the p24{delta}2 protein (a putative receptor for endoplasmic reticulum to Golgi cargo transport) with in each case a change of more than 30-fold (Fig. 5AGo). In black animals adapting to a white background, the expression levels of the 130-kDa complex dropped considerably already after 1 d of adaptation, followed by a slow decrease such that, after 16 d, the expression was not detectable anymore. In contrast, during this adaptation the levels of POMC and p24{delta}2 only decreased gradually (Fig. 5BGo). Thus, during adaptation to a white background, the time course of expression of the 130-kDa complex differed from those of POMC and p24{delta}2.



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  		FIG. 5. Dynamics in the expression of the neuroserpin-containing complex in the intermediate pituitary during background adaptation of Xenopus. A, Fully white-adapted (>21 d; WA) animals were adapted to a black background for 3, 5, 7, 11, 14, or more than 21 d (fully black-adapted; BA). The neurointermediate lobes were isolated and analyzed on Western blot using anti-neuroserpin antibody {alpha}-NS-L. For reference, protein expression of POMC, p24{delta}2 and tubulin was analyzed. B, Fully BA animals were adapted to a white background for 1, 2, 4, 8, 16, or more than 21 d (WA). Experimental conditions as under A. In both A and B, representatives of three independent experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Serine proteases form a large family of intra- and extracellular proteases and have been implicated in a wide variety of biological processes. For example, in the nervous system, proteolysis of the ECM by serine proteases is an important step in the process of synaptic plasticity (29). Obviously, these proteolytic events ask for a delicate control that is provided by regulatory antiproteases called serpins. Neuroserpin is a recently described serpin that is mainly expressed in neuronal and neuroendocrine cells. In this study, the expression and biosynthesis of neuroserpin in the neuroendocrine melanotrope cells of the intermediate pituitary of the amphibian X. laevis were examined. The Xenopus neurointermediate pituitary forms a highly plastic system that can be physiologically manipulated. The plasticity is exhibited by both the innervating hypothalamic neurons and the melanotrope cells, at the levels of proteins, subcellular structures, and synapses (reviewed in Ref.22).

We found that, in the Xenopus melanotrope cells, neuroserpin was initially synthesized as a 44-kDa protein that was subsequently once or twice N-linked glycosylated to products of 47 and 50 kDa, respectively. Glycosylation occurred rapidly after synthesis, as mainly the glycosylated forms were detected during the metabolic cell labeling studies. Both 47- and 50-kDa neuroserpin were secreted into the incubation medium in a slightly modified form, probably as a result of a modification of the N-linked sugar chains, the nature of which remains to be determined. These results constitute the first report on the biosynthesis of neuroserpin under physiological conditions. In the only other biosynthetic study reported thus far, transfected cells were used, and four forms of newly synthesized neuroserpin (45, 47/48, 50, and 55 kDa) were found, of which the 47-/48-, 50-, and 55-kDa forms were glycosylated (21). Previous Western blot analyses of mammalian tissues and transfected cells have revealed that the molecular masses for steady-state neuroserpin range from 40–55 kDa (12, 13, 14, 23). The sizes we detected for immunoreactive Xenopus neuroserpin (44-, 47-/50- and 55-kDa forms) lie within the same range. Because 55-kDa neuroserpin was expressed specifically in the Xenopus pars nervosa, which consists of biosynthetically inactive nerve endings, it was not surprising that we did not detect newly synthesized 55-kDa neuroserpin when the NIL was metabolically labeled. Furthermore, we found the 55-kDa neuroserpin protein in Xenopus brain extracts (data not shown) suggesting that this neuroserpin product represents a neuronal form, whereas 44- and 47-/50-kDa neuroserpin produced in the melanotrope cells of the intermediate pituitary are neuroendocrine forms of the protein. Remarkably, 55-kDa neuroserpin was not N-linked glycosylated and is thus probably otherwise posttranslationally modified. Some serpins are regulated in their antiproteolytic activity by a ligand such as heparin (30). However, heparin-binding seems unlikely for neuroserpin as previous studies have suggested that the antiproteolytic activity of neuroserpin is heparin independent (12).

In addition to 44-, 47-/50-, and 55-kDa neuroserpin, a further immunoreactive product with a molecular mass of 130 kDa was detected. In principle, a neuroserpin multimer may explain the presence of the high-molecular-mass product. Although polymerization of certain serpins is generally caused by mutant forms of the protein (reviewed in Ref.20), serpins may indeed show spontaneous polymerization under physiological conditions (31, 32, 33). However, Xenopus neuroserpin dimer or trimer formation would have resulted in a complex of approximately 100 and approximately 150 kDa, respectively. Because the newly synthesized 130-kDa product was immunoprecipitated only after the 2.5-h chase and not after the 1-h pulse incubation, the occurrence of this product likely reflects the coimmunoprecipitation of neuroserpin with a tightly bound substrate. In general, serpins form a tight complex with their substrate (reviewed in Ref.8). We therefore hypothesize that the 130-kDa neuroserpin-containing product represents an SDS-stable complex of 47-/50-kDa neuroserpin with a substrate. Characterization of the 130-kDa neuroserpin-containing complex using two-dimensional gel electrophoresis revealed that both neuroserpin and the 130-kDa complex are acidic (pI ~5.4). Because the currently known (in vitro) serine protease substrates for neuroserpin (t-PA, u-PA, plasmin, and thrombin) are basic proteins, the observed pI of the complex indicates that these enzymes do not constitute the substrate for Xenopus neuroserpin. Unfortunately, our attempts to further characterize the 130-kDa complex using MALDI-MS and LC/ESI-MS were not successful because of the low endogenous expression levels of the complex. This is not a unique situation. For instance, Misra et al. (34) observed a high-molecular-mass complex containing a serine protease with presumably a serpin, but they could not identify the associated protein. Recently, Drosophila serine protease inhibitor 4 (SP-4), the closest invertebrate ortholog of neuroserpin, has been found to inhibit in vitro the subtisilin-like proprotein convertase furin (35). However, the amino acid sequence of the reactive site of Xenopus neuroserpin (with an arginine and a methionine at positions P1 and P1', respectively) suggests that this serpin will preferably bind a trypsin-like substrate rather than a proprotein convertase such as furin, as the type of target protease is thought to be specified by the positions P1 and P1' of the reactive site (36, 37, 38); an arginine and methionine at P1 and P1', respectively, are also found in chicken neuroserpin, which most likely targets trypsin-like proteases (12). Moreover, in view of the molecular mass of furin (~57 kDa), a neuroserpin-furin complex of 130 kDa would be unlikely. In mouse cortical cultures and embryonic fibroblasts, the very low density lipoprotein receptor and the low density lipoprotein receptor-related protein have been shown to bind and internalize neuroserpin (39). Again, because of their sizes it is not to be expected that the Xenopus very-low-density lipoprotein receptor or lipoprotein receptor-related protein (~100 and ~101 kDa, respectively) will form a 130-kDa complex with Xenopus neuroserpin. Thus, at present the identity of the substrate interacting with Xenopus neuroserpin remains unclear.

Of special interest was the observation that the 130-kDa complex was expressed specifically in the melanotrope cells of the intermediate pituitary and not in the pars nervosa, and that in our biosynthetic studies the complex was not detected in the medium. The complex may thus be formed intracellularly during the transport of neuroserpin through the secretory pathway or in the melanotrope ECM. Furthermore, the complex was found in the melanotropes of only black-adapted and not white-adapted animals, correlating with high and low POMC expression levels, respectively. These findings suggest that the formation of the complex is linked to melanotrope cell activation. The melanotrope cells play a key role in the physiological process of background adaptation of Xenopus. When the animal is on a black background, the melanotrope cells are biosynthetically highly active, producing large amounts of POMC and releasing {alpha}-MSH. On a white background, the cells are inactive, {alpha}-MSH release is inhibited, and the animal turns white. Inhibition of the melanotrope cells is effected by contacting synapses that appear at the ultrastructural level as axon varicosities of neurons originating from the suprachiasmatic nucleus of the hypothalamus (suprachiasmatic melanotrope-inhibiting neurons, SMINs) (40, 41, 42, 43). The terminal varicosities of these inhibitory neurons are much larger and more numerous in white-adapted than in black-adapted animals (more than double the number of active synaptic zones are present on the melanotropes of white- relative to black frogs) (40). When a black animal is placed on a white background, a fast inhibition of melanotrope cell activity is necessary for the animal to shut off {alpha}-MSH secretion instantly. The immediate physiological response (drop of plasma {alpha}-MSH levels) takes place within 30 min after the animal is placed on a white background (44) and may be regulated by the initial release of the neurotransmitter GABA from the SMINs (45). However, to ensure that the melanotrope cells stay inactive during the first few days of adaptation, immediate synaptic changes are also necessary. Obviously, the morphological changes associated with the conversion of the highly active melanotrope cell into a biosynthetically inactive cell take more time (at least 1 wk), which is reflected by only a gradual decrease of POMC expression during such a time period. In black animals adapting to a white background we found a relatively fast decrease in the amount of the 130-kDa neuroserpin-protease complex, possibly through internalization of the complex by the melanotrope cells, implying that the protease and thus proteolytic activity in the melanotrope ECM would be readily available. In melanotropes of white animals adapting to a black background, the 130-kDa complex was detected only after 5 d of adaptation, allowing protease activity in the cell matrix to be still present during the first days of black-background adaptation. Thus, in the first days of adaptation to either a black- or a white background, protease activity is available in the melanotrope ECM and may regulate the formation of the pertinent matrices; for maintenance of the ECM, a proper balance between matrix proteases and their inhibitors is essential (reviewed in Ref.1). Because the ECM is thought to play an important role in synaptic plasticity (reviewed in29, 46, 47, 48), changes in the melanotrope ECM will allow the synaptic plasticity of the hypothalamic neurons and thus enable the dramatic morphological changes that occur in the melanotrope cell during background adaptation of Xenopus.

In conclusion, we report for the first time on the biosynthesis of neuroserpin under physiological conditions, and find that neuroserpin and a 130-kDa neuroserpin-containing complex are up-regulated in the biosynthetically active neuroendocrine melanotrope cells of the intermediate pituitary of black-adapted X. laevis. Because the formation of the 130-kDa complex is linked to melanotrope cell activation, neuroserpin may somehow be involved in the shaping of the melanotrope ECM and thus in the regulation of the synaptic plasticity of hypothalamic neurons that directly innervate this cell.


   Acknowledgments
 
We thank Ron Engels for animal care and Karel Janssen (in memoriam) for technical assistance. We also thank D. A. Lawrence, N. Birch, D. A. Lomas, J. Fransen, and S. Tanaka for providing antibodies.


   Footnotes
 
First Published Online May 19, 2005

Abbreviations: AL, Anterior lobe; Ct, cycle threshold; DDT, dithiothreitol; ECM, extracellular matrix; FENIB, familial encephalopathy with neuroserpin inclusion bodies; LC/ESI-MS, liquid chromatography-electrospray ionization mass spectrometry; m{alpha}-NS, mouse monoclonal IgG against neuroserpin (NS); NIL, neurointermediate lobe; {alpha}-NS-L, rabbit polyclonal IgG against human recombinant neuroserpin; {alpha}-NS-M, rabbit polyclonal IgG against human recombinant neuroserpin; pI, isoelectric point; PMSF, phenylmethylsulfonyl fluoride; POMC, proopiomelanocortin; serpins, serine protease inhibitors; SDS, sodium dodecyl sulfate; t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator.

Received January 27, 2005.

Accepted for publication May 12, 2005.


   References
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 Results
 Discussion
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