Isolation, characterization, and cDNA sequencing of α-1-antiproteinase-like protein from rainbow trout seminal plasma

Isolation, characterization, and cDNA sequencing of

a-1-antiproteinase-like protein from

rainbow trout seminal plasma

Monika Maka,1, Pawel Makb, Mariusz Olczakc, Agata Szalewiczc, Jan Glogowskia,Adam Dubinb, Wieslaw Watorekc, Andrzej Ciereszkoa,*

a Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, PolandbFaculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland

cWroclaw University, Institute of Biochemistry and Molecular Biology, Tamka 2, 50-137 Wroclaw, Poland

Received 22 October 2003; accepted 3 February 2004

Abstract

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-

Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to

homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The

molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and

chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be

a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-

terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop

IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the

reactive loop was significantly similar to carp and mammalian a1-antiproteinases. Both sequences were used to construct primers and obtain

a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that

encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the

similarity to carp a1-antiproteinase and mammalian a1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily

and is similar to a1-antiproteinase.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Fish; Semen; Serpin; Proteinase inhibitor

1. Introduction

Serine proteinase inhibitors (serpins) are a dominant

class of proteinase inhibitors in blood plasma [1]. The

serpin superfamily has evolved over 500 million years with

representatives found in viruses, plants, protozoa, insects,

and higher vertebrates [2]. They are involved in maintaining

homeostasis through multiple regulatory functions, such as

blood coagulation, the complement cascade, fibrynolysis,

protein folding, cell migration, and differentiation, modula-

tion of inflammatory responses, prohormone conversion and

intracellular proteolysis [1]. Serpins account for more than

10% of plasma proteins. The main serine proteinase inhib-

itor in blood is a1-proteinase inhibitor (a1-PI), also called

a1-antiproteinase or a1-antitrypsin. This inhibitor has a

broad specificity towards serine proteinases, but neutrophil

elastase seems to be its physiological target [3].

In contrast to blood, seminal plasma of teleost fish is

characterized by a very low (1–3 mg/ml) protein concen-

tration. We have found previously [4] that fish seminal

plasma exhibits a distinct antitrypsin activity. Further elec-

trophoretic studies revealed that there are a number of

species-specific antitrypsin bands in seminal plasma with

different affinity towards cod and bovine trypsin [5]. Our

0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2004.02.001

* Corresponding author. Tel.: +48-89-535-7426; fax: +48-89-535-

7421.

E-mail address: [email protected] (A. Ciereszko).1 Present address: Department of Immunology, Collegium Medicum,

Jagiellonian University, Czysta 18, 31–121 Krakow, Poland.

www.bba-direct.com

Biochimica et Biophysica Acta 1671 (2004) 93–105

results indicated that, opposite to mammals and birds, most

serine proteinase inhibitors of fish seminal plasma are of

high molecular weight and are related to blood plasma

inhibitors. This suggests that they may belong to the serpin

family. The presence of serpins in fish seminal plasma has

been indicated by Huang et al. [6]. These authors found that

the proteinase inhibitor of the serpin family (p62), isolated

from carp perimeningeal fluid, is also present in numerous

tissues, including milt. This protein has been cloned and

recognized as the fish equivalent of a1-antitrypsin [7].

However, according to our knowledge, isolation and puri-

fication of serine proteinase inhibitors from fish seminal

plasma has never been attempted.

We have found recently that the antitrypsin activity of

rainbow trout seminal plasma can be sequentially resolved

into three fractions (I–III) after ion-exchange chromatogra-

phy on Q-Sepharose [8]. Antiproteinase of fraction II

(inhibitor II) can be detected in seminal and blood plasma

using cod and bovine trypsin and chymotrypsin. In the

current study, we report the purification and physicochem-

ical and kinetic characterization of this inhibitor. The N-

terminal and reactive loop amino acid sequences have also

been obtained. Using information derived from these

sequences a cDNA from rainbow trout liver was obtained.

Analysis of characteristics of purified protein and the cDNA

sequence demonstrate that properties of this inhibitor are

similar to a1-proteinase from carp perimeningeal fluid.

2. Materials and methods

2.1. Source of milt

Milt was obtained by stripping rainbow trout (Oncorhyn-

nchus mykiss Walbaum, 1792) maintained at the Rutki

Salmonid Research Laboratory, Institute of Inland Fisheries,

Olsztyn, Poland. Seminal plasma was obtained by centrifu-

gation (10,000� g, 10 min, 4 jC) and stored at � 20 jC.

2.2. Determination of protein

Protein concentration was determined by the method of

Bradford [9] using Sigma reagents and bovine serum

albumin as a standard.

2.3. Polyacrylamide gel electrophoresis (PAGE)

Slab gel native (PAGE) and sodium dodecyl sulfate

(SDS-PAGE) electrophoresis were conducted according to

the method of Laemmli [10] using the Mighty Small II

electrophoresis system (Hoefer-Amersham Biosciences, Pis-

cataway, NJ, USA) as was described in the manufacturer’s

instructions. Antitrypsin activity after PAGE was identified

in gels using bovine and cod trypsin or chymotrypsin,

according to Uriel and Berges [11]. Proteins in gels were

stained with Coomassie Brilliant Blue R-250. Molecular

weights of proteins were estimated with the use of the

Kodak 1D program (Eastman Kodak Company, New Ha-

ven, USA).

2.4. Measurement of antitrypsin activity

Antitrypsin activity was measured by the inhibition of

cod trypsin amidase according to the method of Geiger and

Fritz [12] with modifications previously described [5].

2.5. Purification of inhibitor from seminal plasma

Seminal plasma was dialyzed for 24 h against 20 mM

Tris–HCl buffer pH 7.6, (buffer A) at 4 jC. Ion-exchange

chromatography was carried out as previously described

[8]. Briefly, the dialysate was centrifuged and applied to a

column X/K 16/10 of HiLOad Q-Sepharose connected to a

FPLC system (Amersham Biosciences, Uppsala, Sweden).

The column was equilibrated with buffer A. All antitrypsin

activity was bound under these conditions. The bound

proteins were eluted with a linear NaCl gradient (0–0.5

M) in buffer A at a flow rate of 60 ml/h. Three fractions of

antiproteinase activity were obtained as described previ-

ously [8], eluted at a conductivity range of 1.2–1.4, 13.1–

14.4, and 14.8–17.6 mS. The second fraction was pooled

and lyophilized. The lyophilisate was diluted in 2 ml H2O

and applied to a column X/K HiLoad 16/60 of Superdex

200 equilibrated with 50 mM Tris–HCl buffer (pH 7.6).

Elution was conducted with the same buffer at a flow rate

of 60 ml/h. Fractions of the pure inhibitor were lyophi-

lized, then resuspended in H2O and dialyzed against 20

mM Tris–HCl buffer, pH 7.6. The preparation was stored

at � 20 jC.

2.6. Determination of molecular weight

Molecular weight of the native inhibitor was estimated

using gel filtration in an X/K HiLoad 16/60 Superdex 200

column (Amersham Biosciences) equilibrated with 0.05 M

Tris–HCl buffer (pH 7.6) containing 0.15 M NaCl. The

column was calibrated with low and high molecular weight

protein standards (Amersham Biosciences).

The molecular weight of denatured inhibitor was estima-

ted by 12.5% SDS-PAGE slab gels. A low molecular weight

kit 14–94 kDa supplied by Amersham Biosciences was

used for calibration of gels. Molecular weights of protei-

nases and proteinase inhibitors were estimated with the use

of the Kodak 1D program (Eastman Kodak Company).

2.7. Complex formation between the inhibitor with cod and

bovine trypsin

The inhibitor (4 Ag) was incubated in 20 mM Tris–HCl

buffer (pH 7.6) for 15 min with a molar excess of either cod

or bovine trypsin. The samples were then boiled in the

presence of SDS and analyzed using SDS-PAGE.

M. Mak et al. / Biochimica et Biophysica Acta 1671 (2004) 93–10594

2.8. Determination of isoelectric point

Chromatofocusing was performed using 0.5� 20 cm of

PBEk94 column (Amersham Biosciences) equilibrated

with 0.025 M imidazole-HCl buffer (pH 7.4). Elution was

performed using 8-fold diluted Polybuffer 74-HCl (pH 5.0).

Isoelectrofocusing of proteinase inhibitor was carried out in

ready IEF gels pH 5–8 using a Ready Gels Cell with

cathode and anode IEF buffers (Bio-Rad, Hercules, CA,

USA). The isoelectric Focusing Marker Kit (Sigma, St.

Louis, MO, USA) was used for calibration of gels.

2.9. Interaction with lectins

Interaction of the inhibitor with the following digoxige-

nin-labeled lectins were tested: Galanthus nivalis agglutinin

(GNA) with affinity for terminal D-mannosyl residues in the

high mannose or hybrid type oligosaccharides; Sambucus

nigra agglutinin (SNA) with affinity for sialic acid bound

via a(2–6) to D-galactose or N-acetyl-D-galactosamine;

Maackia amurensis agglutinin (MAA) with affinity for

sialic acid bound via a(2–3) to D-galactose; Datura stra-

monium agglutinin (DSA) with affinity for terminal D-

galactose h(1–4) N-acetyl-D-glucosamine in complex or

hybrid glycans and peanut agglutinin (PNA) indicating

galactose-h(1–4) N-acetylgalactosamine (the disaccharide

usually forming core the unit of O-glycans). The inhibitor

(1 Ag) was directly bound to nitrocellulose (NC) mem-

branes using the dot-blot method. The blots were then

blocked and incubated with lectins according to the proce-

dure described in the DIG Glycan Differentiation Kit

(Boehringer, Mannheim, Germany) with slight modifica-

tions concerning blocking procedure and lectin detection

(0.25% gelatin in tris-buffered saline (TBS) and horseradish

peroxidase (HRP)-labeled Fab fragments of sheep anti-

digoxygenin antibodies were used, respectively). Carboxy-

peptidase Y, transferrin, fetuin, and asialofetuin were used

as control proteins. The enzyme conjugated probes were

visualized by peroxidase reaction using 0.06% 4-chloro-1-

naphtol in 50 mM Tris–HCl (pH 7.6) containing 0.01%

H2O2. Reactivity with ConA was tested on blots by the

ConA-HRP method according to Olden and Yamada [13]

and Clegg [14]. Activity of HRP on blots was detected

using 4-chloro-1-naphtol (0.06%) in 50 mM Tris–HCl

buffer (pH 7.5) containing 0.01% H2O2.

2.10. Enzymatic deglycosylation

Enzymatic deglycosylation of denatured inhibitor was

performed with N-glycosidase F (PNGase F, EC 3.2.2.18)

from Flavobacterium meningosepticum (Oxford Glycosys-

tem, UK). Inhibitor II (6 Ag) dissolved in the 20 mM

sodium phosphate buffer (pH 7.5) containing 50 mM

EDTA and 0.5% SDS was denatured by boiling for 2

min. Then n-octylglucoside was added to a final concen-

tration of 2%, followed by PNGase F in the proportion 25

mU of endoglycosidase activity/mg of protein. In the

control sample PNGase F was omitted. The reaction was

carried out at 37 jC for 18 h. The final reaction products

were analyzed by SDS-PAGE. Time-dependent deglycosy-

lation of inhibitor II was carried out under the same

conditions. At indicated time intervals equal aliquots were

withdrawn and the reaction was stopped by heat denatur-

ation in the SDS-PAGE sample buffer. The intermediate

products were then analyzed by SDS-PAGE. The effective-

ness of deglycosylation was checked by the reactivity with

ConA. For this purpose, samples resolved in SDS-PAGE

were transferred onto NC membranes using 25 mM Tris-

192 mM glycine/20% (v/v) methanol buffer (pH 8.3)

according to Towbin et al. [15]. Electrotransfer was per-

formed for 1 h at 200 mA in a Fastblot B33 semidry

apparatus (Biometra, Goettingen, Germany). Glycoproteins

bound to nitrocellulose were detected using the ConA-HRP

method as described earlier. Molecular weights of native

and deglycosylated inhibitor II were estimated with the use

of the Kodak 1D program.

2.11. Measurement of association rate constant

Concentrations of active bovine trypsin, cod trypsin

(Sigma), and bovine chymotrypsin (Sigma), were deter-

mined by active-site titration with p-nitrophenyl-pV-guani-

dinebenzoate [16]. Porcine elastase (ICN Biomedicals, Inc.,

Costa Mesa, USA) was titrated against a standardized

solution of human a-1-proteinase inhibitor as a secondary

standard using MeO-Suc-Ala-Ala-Pro-Val-pNA (Sigma) for

measurement of residual enzyme activity. The concentration

of active trypsin inhibitor from rainbow trout seminal

plasma was determined with the use of a standardized

solution of bovine trypsin, using Bz-Arg-pNA (BAPNA)

as substrate. The second-order association rate constants of

the inhibitor with various serine proteinases were deter-

mined by the method of Bieth [17]. Briefly, equimolar

mixtures of enzyme and inhibitor were incubated for in-

creasing times at room temperature in 0.2 M Tris–HCl

buffer (pH 8.0). Residual activities of enzymes were mea-

sured with the following proteinase substrates: BAPNA

(trypsin), Suc-Ala-Ala-Pro-Phe-pNA (chymotrypsin), and

MeO-Suc-Ala-Ala-Pro-Val-pNA (elastase). These substrates

were purchased from KabiVitrum, Stockholm, Sweden. The

association rate constant for each enzyme and inhibitor as

well as delay times of inhibition were calculated according

formulas of Bieth [17]. The final inhibitor II concentration

used in calculations was 116 nM and was equivalent to a

final inhibitor concentration in plasma calculated from the

purification table.

2.12. Amino acid sequence analysis

The N-terminal amino acid sequence analysis was per-

formed on a gas-phase sequencer (Model 491, Perkin

Elmer-Applied Biosystems, Foster City, CA, USA) at the

M. Mak et al. / Biochimica et Biophysica Acta 1671 (2004) 93–105 95

BioCenter (Jagiellonian University, Krakow, Poland). The

phenylthiohydantoin derivatives were analyzed by on-line

gradient high performance liquid chromatography on a

microgradient delivery system model 140C equipped with

programmable absorbance detector model 785A (both from

Perkin Elmer-Applied Biosystems). Sequence comparisons

were performed using the database SWISS-PROT (http://

www.ncbi.nlm.nih.gov/blast).

2.13. Determination of N-terminal sequence of the inhibitor

Inhibitor II (1 nM) was desalted by HPLC using reversed

phase C-18 column (Waters, Milford, USA.) equilibrated

with 0.1% trifluoroacetic acid. Inhibitor II was eluted by a

gradient of 0.07% trifluoroacetic acid with 80% acetonitrile.

Eluted inhibitor was lyophilized, dissolved in water, and

sequenced.

2.14. Determination of sequence of peptide from reactive

center obtained after cleavage at P1–P1V

Inhibitor II was incubated for 15 min with bovine trypsin

at a molar ratio of 1.3:1.0 in order to obtain peptide from the

reactive center. After incubation, the residual trypsin activity

was inhibited by 4 mM of synthetic inhibitor PVBLOC

(Boehringer). Samples were boiled and subjected to tris-

tricine SDS-PAGE [18] and electroblotted onto PVDF mem-

brane using 10 mM 3-(cyclohexylamino)-1-propanesulfo-

noic acid-NaOH (pH 11.0) containing 10% methanol. The

membrane was stained in 0.1% CBB in 40% methanol, 1%

acetic acid and destained in 50% methanol. The band of a 3

kDa peptide was cut off and sequenced.

2.15. Determination of sequence of peptide from reactive

center obtained after cleavage at P4

Inhibitor II cleaved above P1–P1V was obtained after

incubation of the inhibitor with proteinase V8 from Staph-

ylococcus aureus at a mass ratio of 200:1. The reaction was

stopped with PVBLOC when residual inhibitory activity

was approximately 20% in order to avoid further proteolysis

of other than the exposed reactive loop sites of the inhibitor.

Further steps including electrophoresis, electroblotting, and

sequencing were performed as described above.

2.16. cDNA isolation from rainbow trout liver and sequence

analysis

Fresh liver tissue was immediately frozen in liquid

nitrogen and stored at � 80 jC. The tissue was homoge-

nized using a mortar and pestle followed by QIAShredder

microcolumns (Qiagen GmbH, Hilden, Germany). Total

RNA from 40 mg of homogenized tissue was purified using

the RNeasy Mini Kit (Qiagen) as recommended by the

manufacturer. The integrity of the total RNA was checked

with formaldehyde agarose electrophoresis.

To identify the cDNA of the inhibitor we designed a set of

degenerate PCR primers, based on the sequence from the N-

terminus of the protein and sequences of the peptides

generated by enzymatic digestion. A pair of forward 5V-

CA(CT)CA(CT)AT(ACT)GCIGGIGA(AG)GC-3V and re-

verse 5V-TT(ACGT)AGCATIATIGT(AG)TC(ACGT)GG-3V

primers gave a discrete PCR product of 1085 bp. The product

was cloned into pCR4-TOPO vector (Invitrogen Corpora-

tion, Carlsbad, CA, USA) and sequenced. The full-length

cDNA was further synthesized using the GeneRacer Kit

(Invitrogen) according to the manufacturer’s protocol. For

5VRACE and 3VRACE, a reverse 5V-GGTGTTGGCTTTGC-

TCAGGCTCCTG-3Vprimer, and a forward 5V-TTACCCA-

CAAGGCTGTGCTGAGCG-3V primer were designed,

respectively. Reverse transcription was carried out at 55 jC

for 1 h, using the Thermoscript reverse trancriptase (Invi-

trogen). All PCR reactions were performed in the presence of

Dynazyme Ext Polymerase (Finnzymes Oy, Espoo, Finland).

The same conditions of touchdown PCR were applied to

amplify the 5Vand 3VRACE products: initial incubation at 94

jC for 3 min was followed by five cycles of denaturation (94

jC, 30 s), annealing and extension (72 jC, 60 s), then five

cycles of denaturation (94 jC, 30 s), annealing and extension

(70 jC, 60 s), then 25 cycles of denaturation (94 jC, 30 s),

annealing (65 jC, 30 s) and extension (72 jC, 30 s), and

finally 10 min at 72 jC. The reaction volume was 50 Al. The

5Vand 3VPCR products were cloned into pCR4-TOPO vector

and sequenced. Motif scan in protein sequence was per-

formed using the Prosite database (http://us.expasy.org/

prosite/) according to Falquet et al. [19]. The putative

signal peptide was determined using SignalP V1.1 (http://

www.cbs.dtu.dk/services/SignalP) according to Nielson et

al. [20]. Molecular weight and isoelectric point of the

translated protein was calculated using ProtParm Tool

program (http://us.expasy.org/cgi-bin/protparam).

3. Results

3.1. Isolation of inhibitor

The two-step isolation procedure led to a 73-fold purified

inhibitor with a yield of about 20% (Table 1). During ion-

exchange chromatography the inhibitor was eluted before

two main plasma proteins and for this reason it was possible

Table 1

Isolation of inhibitor II from rainbow trout seminal plasma

Fraction Total

protein

(mg)

Total

activity (U)*

Specific

activity

(U mg� 1)

Fold Yield

(%)

Seminal plasma 14.1 2.728 0.19 1 100.0

Q-Sepharose 0.11 0.654 5.94 31.3 23.9

Superdex 200 0.04 0.558 13.95 73.4 20.4

*U—one unit was defined as inhibitor amount that inhibits activity of

one international enzymatic unit of cod trypsin.


http:\\www.ncbi.nlm.nih.gov\blast

http:\\us.expasy.org\prosite\

http:\\www.cbs.dtu.dk\services\SignalP

http:\\us.expasy.org\cgi-bin\protparam

to obtain relatively pure preparations after this step (Fig. 1).

Final purification has been achieved using gel filtration. The

inhibitor migrated during PAGE and SDS-PAGE as a single

band. It could be visualized after PAGE with the zymogram

method using either cod trypsin, bovine trypsin, or bovine

chymotrypsin.

3.2. Physicochemical characteristics

The molecular weight of the inhibitor, estimated using gel

filtration and SDS-PAGE (in reducing and non-reducing conditions) was determined to be 56 kDa. The inhibitor

formed SDS-stable complexes with bovine and cod trypsin

(Fig. 2). Molecular weights of the inhibitor, bovine trypsin,

cod trypsin, inhibitor/bovine trypsin complex, and inhibitor/

cod trypsin complex were determined to be 56, 22.3, 21.6,

65, and 66 kDa, respectively. Two bands of lower molecular

weight were also seen which suggests proteolysis of the

Fig. 4. Deglycosylation of inhibitor II. Lane 1—Molecular weight

standards; lane 2—inhibitor II, control sample; lane 3—18 h incubation

of inhibitor II with PNGase F. (A) CBB staining of the gel, (B) ConA-HRP

staining of NC membrane.

Fig. 3. Isoelectric focusing of inhibitor II. Isoelectrofocusing of proteinase

inhibitor was carried out using ready IEF gels (pH 5–8) in the Ready Gels

Cell and cathode and anode IEF buffers (Bio-Rad). Isoelectric Focusing

Marker Kit (Sigma) was used for calibration of gels. (1) Carbonic

anhydrase I, carbonic anhydrase II (pI 6.6, pI 5.9); (3) methyl red (pI 3.8);

(4) h-lactoglobulin (pI 5.1); (2) inhibitor II (pI 5.42).

Fig. 2. Formation of SDS-stable complexes between inhibitor II and cod

and bovine trypsin. The inhibitor (4 Ag) was incubated in 20 mM Tris–HCl

buffer (pH 7.6) for 15 min with a molar excess of either cod or bovine

trypsin. Samples were then boiled in the presence of SDS and analyzed

using SDS-PAGE. (1) Molecular weight standards; (2) bovine trypsin; (3)

cod trypsin; (4) inhibitor II; (5) complex inhibitor II/bovine trypsin; (6)

complex inhibitor II/cod trypsin.

Fig. 1. Electrophoretic profiles of fraction II after HiLoad Q-Sepharose. A

column was equilibrated with 20 mM Tris–HCl buffer and proteins were

eluted using a linear NaCl gradient (0.0–0.5 M). Fractions 109–115 were

collected and further purified using gel filtration.


complexes. During chromatofocusing the inhibitor was elut-

ed at pH 5.43. The isoelectric point estimated by electro-

focusing was determined to be 5.42 (Fig. 3).

3.3. Interaction with specific lectins

The glycoprotein character of inhibitor II was first shown

by a positive reaction with ConA. Using digoxigenin-

labeled lectins, an intense reaction of the inhibitor with

DSA and MAA, a weak reaction with SNA, and no reaction

with GNA and PNA were observed (data not shown). This

result indicates the presence of complex glycans, with

branches both sialylated and with non-sialytated. Based

on the intense reaction with MAA and a weak reaction

with SAA, the sialic acid appeared to be mainly a(2–3)

terminally linked to galactose and to a lesser extent linked

a(2–6) to galactose or N-acetylgalactosamine. Inhibitor II

does not contain high mannose and hybrid glycans as was

evidenced by the negative reaction with GNA. The negative

reaction with PNA indicates the lack of O-glycosidically

linked glycans.

3.4. Estimation of carbohydrate content based on deglyco-

sylation of the inhibitor

After deglycosylation with PNGase F the molecular

weight of inhibitor II decreased to about 47 kDa as shown

in SDS-PAGE (Fig. 4A). Deglycosylated inhibitor II did not

react with ConA which indicates that a complete deglyco-

sylation occurred (see Fig. 4B) Thus, inhibitor II contains

about 16% (w/w) carbohydrate.

3.5. Time-dependent deglycosylation

SDS-PAGE analysis of the inhibitor subjected to degly-

cosylation revealed the presence of one intermediate of

molecular weight 50.5 kDa appearing within the first hour

of PNGase F treatment (Fig. 5A). As shown by its reactivity

with ConA, this modified protein was still glycosylated

(Fig. 5B). A completely deglycosylated protein was ob-

served only after 24 h of deglycosylation, forming a single

protein band, non-reactive with ConA (Fig. 5). Thus, the

sugar moiety of the inhibitor contains at least two N-linked

oligosaccharide chains.

3.6. Association rate constant

The percentage of active enzymes and inhibitor were as

follows: cod trypsin 58%, bovine trypsin 65%, bovine

chymotrypsin 56%, human elastase 59%, inhibitor II 74%.

Titrated enzymes and inhibitor were used for association rate

constants determination. The obtained constants values were

used for delay times calculations, i.e. the time required for

complete inhibition of proteinase in vivo (see Table 2).

3.7. Amino acid sequencing

N-terminal sequencing identified the following 30 amino

acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG.

After complex formation of inhibitor II with trypsin the

following 21 amino acids of released peptide (P1V–P21V)

were identified: SLPDTIMLNRPFLLFILEDST. Sequenc-

ing of the peptide obtained after cleavage with proteinase

V8 produced the following 10 amino acids IMPMSLPDTI.

It appeared that proteinase V8 cleaved inhibitor II at P4

(Fig. 6).

Fig. 5. Time-dependent deglycosylation. Lanes 1–7: samples of purified inhibitor II after 0, 15, 30 min, 2, 4, 6, and 24 h of PNGase F treatment. S—molecular

weight standards (see Fig. 4). (A) CBB staining of the gel, (B) ConA-HRP staining of NC membrane.

Table 2

Association rate constants and inhibition delay times for inhibitor II

Proteinase kass (M� 1 s� 1) t (s)

Cod trypsin 2� 106 20

Bovine trypsin 11�106 4

Bovine chymotrypsin 4� 106 10

Porcine elastase 1�105 430Fig. 6. Cleavage of inhibitor II with trypsin and proteinase V8.


3.8. cDNA cloning

The nucleotide sequence and the deduced amino acid

sequence are shown in Fig. 7. A putative ATG codon is

located at nucleotide 33. The open reading frame is pre-

dicted to encode a protein of 426 amino acids with a

calculated molecular weight of 47,529 Da and pI 5.86. Both

peptide sequences determined by Edman sequencing

matched perfectly to the amino acid sequence deduced from

the analyzed cDNA. Hydrolysis of a putative signal peptide

should produce mature protein containing 407 amino acids

with a calculated molecular weight of 45,520.5 Da and pI

Fig. 7. Nucleotide sequence and deduced amino acid sequence of a1-antiproteinase-like protein. Sequences determined by amino acid sequence analysis are

underlined and attachment sites for N-linked sugar chains are double underlined. The sequence has been deposited in EMBL Nucleotide Sequence Database

with accession number AJ558113.


5.87. However, the N-terminal sequence obtained by amino

acid sequence analysis lacked two first amino acids in

comparison with a putative N-terminal sequence deduced

from cDNA. Motif search of the sequence revealed the

presence of the sequence IMLNRPFLLFI at P6V–16V that

has been identified as the ‘‘serpine signature’’ PROSITE

PDOC00256 (LIVMFU)-x-(LIVMFYAC)-(DNQ)-

(RKHQS)-(PST)-F-(LIVMFY)-(LIVMFYC)-x-(LIVM-

FAH). A histidine-rich region (PROSITE PDOC50099) has

been also localized at the N-terminal region. Two potential

N-glycosylation sites Asn267 and Asn279 (PROSITE

PDOC 00001) have been detected between 267–269

(NFT) and 279–282 (NSS). Five putative PKC phosphor-

ylation sites (PROSITE PDOC00005, pos. 235–237, 256–

258, 365–367, 410–412, 424–426) and CK2-phosphory-

lation sites (PROSITE PDOC00006, pos. 49–52, 314–317,

330–333, 383–386, 391–394) were identified. Potential N-

mirystyolation sites (PROSITE PDOC00008) have been

localized at pos. 3–8, 90–95, 147–152, 339–344, and

376 – 381 and a tyrosine sulfation site (PROSITE

PDOC00003) at pos. 255–269.

4. Discussion

According to our knowledge, this is the first report

concerning the isolation and characterization of a serine

proteinase inhibitor from fish seminal plasma. Isolation of

the inhibitor from rainbow trout seminal plasma was

achieved by sequential application of Q-Sepharose ion-

exchange chromatography and Superdex 200 gel filtration.

This procedure is much simpler and faster in comparison

with protocols employed for purification of proteinase inhib-

itors from fish blood or perimeningeal fluid [6,21]. Seminal

plasma of rainbow trout, like most teleost fish, is character-

ized by a very low protein concentration (1–3 mg/ml). The

inhibitor is one of the main proteins of seminal plasma (it can

be detected directly by CBB-staining in seminal plasma). Q-

Sepharose chromatography was very efficient and produced

the inhibitor preparations with a high purity.

The molecular weight of the isolated inhibitor was deter-

mined to be 56 kDa. This value was obtained using both gel

filtration and SDS-PAGE in reducing and non-reducing

conditions. Therefore, it may be concluded that the inhibitor

is composed of a single polypeptide chain. Rainbow trout

inhibitor has a similar weight to the a1-proteinase inhibitor

of carp serum (55 kDa, [22]) and a lower weight than carp

perimeningeal fluid serine proteinase inhibitor CP9 (62 kDa;

[6]). The molecular weight calculated from nucleotide se-

quencing (45.5 kDa) is in close agreement with the molec-

ular weight obtained after deglycosylation (47 kDa).

The isoelectric point of the inhibitor from rainbow trout

seminal plasma (pI = 5.4) is similar to values described for

other fish serpins, for example pI of salmon antithrombin

is within the range of 4.7–6.0 [23] but lower values of pI

(4.2–4.9) have been reported for mammalian a1-proteinase

inhibitor [3,24–27]. It should be noted that many mam-

malian a1-proteinases show microheterogeneity and multi-

ple isoforms can be visualized by isoelectric focusing

[3,27]. Our data indicate that inhibitor II is present as a

single band after isoelectric focusing (Fig. 4). In this

respect, inhibitor II may resemble horse a1-proteinase

[3]. The isoelectric point calculated from the amino acid

sequence (5.87) was higher than the pI of a native protein.

It is likely that components of a carbohydrate moiety, such

as sialic acid residues, contribute to a lower pI of the

native protein.

Inhibitor II appeared to be a glycoprotein and its carbo-

hydrate content falls within the range characteristic for

serpins (10–20%), including a1-antiproteinase [3]. Treat-

ment with PNGase F caused a complete deglycosylation of

inhibitor II. This indicates the presence of N-linked carbo-

hydrate side chains in the molecule of inhibitor II. Such

presence is a characteristic feature of most a1-antiprotei-

nases and carbohydrate side chains are N-glycosidically

linked to the Asn side chains [3]. N-linked but not O-linked

carbohydrate side chains were also found in serpin p62 of

carp perimeningeal fluid [6]. Binding of specific lectins to

the carbohydrate moieties indicates that N-linked carbohy-

drates of inhibitor II are sialylated, complex oligosacchar-

ides. The sialic acid appeared to be terminally linked mainly

through a(2–3) and to a lesser extent through a(2–6) to

galactose based on the intense reaction with M. amurensis

agglutinin and a weak reaction with S. nigra agglutinins. A

Fig. 7 (continued ).


positive reaction with D. stramonium agglutinin suggests

the presence of terminal Galh(1–4)GlcNAc. This may

suggest that a partial loss of terminal sialic acid occurred

leaving some branches terminated by Galh(1–4)GlcNAc, as

was suggested for salmon antithrombin [23]. Failure of

inhibitor II to react with G. nivalis agglutinin suggests that

it does not contain N-linked, high mannose glycan. Overall,

our data indicated that inhibitor II is a glycoprotein and its

carbohydrate components may have a similar structure to

a1-antiproteinase. Analysis of cDNA revealed a presence of

two potential glycosylation sites (Fig. 7). This corresponds

well to the results of time-dependent deglycosylation of

inhibitor II that showed two oligosaccharide chains present

in native protein. Two glycosylation sites of rainbow trout

inhibitor are located at the same place as in a1-antiprotei-

nase of carp (Fig. 8).

The presence of multiple potential phosphorylation and

mirystyolation sites and the tyrosine sulfation site suggests

that rainbow trout a1-antiproteinase could be modified by

these post-translational modifications. Such modifications

have been described for some serpins, for example tyrosine

sulfation for heparin cofactor II [28] and tyrosine phosphor-

ylation for maspin [29]. It has been postulated that phos-

phorylation may be important modulator of biological

functions of uterine serpins [30]. On the other hand, no

phosphorylation has been found in some serpins in spite of

the presence of multiple phosphorylation sites [31]. Further

studies are necessary to explain if the above mentioned sites

of potential post-translational modifications are indeed uti-

lized in rainbow trout a1-antiproteinase.

Purified inhibitor formed SDS-stable complexes with

bovine and cod trypsin, a unique feature of serpins. Molec-

ular weights of complexes appeared to be lower than the

sum of the individual components. A similar phenomenon

was also observed for a1-proteinase inhibitor from carp

blood plasma [21] and rabbit plasma a1-antiproteinase [32].

Fig. 8. Homology alignment of rainbow trout (first row) and carp (second row) a1-AP. The same amino acids are marked in bold. Stars denote amino acids

strictly conserved in >70% of serpin sequences [2]. N-glycosylation sites are double underlined.


It is likely that the lower molecular weight of the com-

plexes were produced due to a partial degradation of the

inhibitor or trypsin. This phenomenon may be partially

attributed to the loss of the 4 kDa C-terminal peptide from

the reactive loop of the inhibitor. Such a peptide was

observed during complex formation between of the inhib-

itor with trypsin and was used for amino acid sequencing.

Since molecular weights of the complexes were lower by

12–13 kDa, this suggests that further proteolytic degrada-

tion of the complexes occurred. It is known that some

serpin-proteinase complexes show increased susceptibility

to cleavage by residual free enzyme [33]. Presumably the

incubation time employed in our experiment (15 min) was

too long and shorter periods are necessary to obtain non-

degraded complexes. Further studies aimed to test complex

formation in relation to time of incubation should clarify

this issue.

Cod trypsin, bovine trypsin and chymotrypsin could be

used for visualization of the inhibitor purified in the present

work. Data of kinetic analysis (Table 2) confirmed that the

inhibitor efficiently inhibits all of these three serine protei-

nases, giving a delay time (t) toward these enzymes in the

range of seconds to tens of seconds. Delay time is a kinetic

parameter that is five times greater than half-life time and

represents a period of time needed for almost complete

inhibition of enzyme activity. Obtained values testify that

the inhibition of trypsin and chymotrypsin is therefore fast

and effective. Obtained results agree with the data of

Aranishi [21] who found that a1-antiproteinase of carp

blood plasma inhibited bovine trypsin and chymotrypsin.

On the other hand, the inhibitor of rainbow trout seminal

plasma was less effective against elastase in comparison to

trypsin and chymotrypsin. Lower affinity towards elastase

may be a characteristic feature of this inhibitor, opposite to

mammalian a1-antiproteinases that have a high affinity

towards elastase and its biological role in humans is defined

by control of leukocyte elastase [34].

Although no statistically significant homology between

N-terminal sequence of inhibitor II obtained by amino acid

sequencing and any known protein was found, an arbitrary

alignment suggested that there is some similarity to N-

terminal sequence of a homolog of serine proteinase inhib-

itor (CP9) isolated from common carp perimeningeal fluid

[7], as is shown in Fig. 8. This is in agreement with a rule

that amino-terminal regions of serpins are most variable [3].

It is known, however, that the sequence within the reactive

site region of serine proteinase inhibitors is highly con-

served [35]. The amino acid sequence within the reactive

center of inhibitor II showed a high degree of homology

with vertebrate a1-antiproteinases, including: 76% identity

with both a1-antitrypsin homolog precursor and serine

proteinase inhibitor CP9 from common carp perimeningeal

fluid [7], 70% identity with rabbit a1-antitrypsin F precursor

[36], 60% identity with a1-antiproteinase of the African

clawed frog (Xenopus leavis, [37]), 54% identity with

guinea pig a1-antiproteinase S precursor [35], and 52%

with ground squirrel a-antitrypsin-like protein [38] and

horse a1-antitrypsin (accession no., AAC83412).

The reactive site region of the a1-antiproteinase family

can be classified into orthodox and unorthodox types [35].

The orthodox type has the P3–P3V consensus sequence of

Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Val, Leu, Ileu or

Met. These amino acids are regarded to be very similar in

molecular size and shape as well as in hydrophobicity, and

frequently interchange during evolution of many protein

families. Met at the P1 site is believed to be crucial in the

physiological activity of the orthodox a1-antiproteinases.

The P3–P3V sequence of inhibitor II is Met-Pro-Met-Ser-

Leu-Pro (Fig. 8). This agrees with the consensus of the

orthodox type. Due to the presence of Met at P1 mammalian

a1-antiproteinases are inactivated by oxidation [3] and a

similar phenomenon is expected to be true for fish a1-APs.

The sequences of the peptides obtained from the enzy-

matic digestion aligned perfectly to the deduced amino acid

sequence drawn from the mRNA isolated from the liver as

was anticipated based on our previous results indicating the

same electrophoretical and chromatographical behavior of

inhibitor II from blood and seminal plasma. The highest

homology alignments were found with antitrypsins from

carp (61% and 63%) and African frog (48%). Moreover,

numerous significant alignments were detected with 39–

43% identities with mammalian a1-antiproteinases. This

strongly suggests that rainbow trout serpin described in this

study belongs, similarly to carp antitrypsin, to clade ‘‘a’’ of

serpins (antitrypsin-like) as was classified by Irving et al. [2].

Putative conserved serpin domains have been identified

by BLAST program (Fig. 9). Moreover, most (44 of 51, Fig.

8) of strictly conserved amino acids in >70% of the serpin

sequences are found in both carp and rainbow trout serpins.

These amino acids are located in the sites important for

serpin conformation and their significance has been reviewed

by Irving et al. [2]. These regions include the hinge (P15–P9,

Fig. 9), located within the ‘‘serpin signature’’, that provides

mobility essential for the conformation change of the reactive

center loop (RCL); the breach and the shutter located on the

top and near the center of the A h-sheet, respectively; and the

gate, including strands s3C and s4C. The breach and shutter

facilitate sheet opening and accept the conserved hinge of the

RCL as it inserts through the gate. Preservation of critical

amino acids in the serpins likely contributes to the conser-

vation of their structure, despite the evolutionary distances

among serpins and their different functions.

A statistically significant histidine-rich motif has been

found between positions 22 and 62 using the program of

Falquet et al. [19]. Interestingly, histidine-rich motifs have

been identified in serpin pNiXa isolated from Xenopus

laevis. It was shown that this motif is involved in binding

of nickel ions [39,40]. Serpin pNiXa [41] is present in

oocytes and embryos and is engaged in the induction of

maturation of oocytes and may regulate proteolytic process-

es during embryonic development [40]. This serpin shows

38% homology to rainbow trout a1-antiproteinase described


in this study and is also produced by the liver. A histidine-

rich domain is also present in a1-antiproteinase of X. laevis

([37], showing 48% homology with trout a1-antiproteinase).

The histidine-rich region can be also seen in the N-terminal

part of carp a1-antiproteinase. Altogether these results

suggest that the presence of a histidine-rich motif may be

a characteristic feature of serpins of lower vertebrates.

Similar to the majority of the mammalian a1-antiprotei-

nases, a mature rainbow trout a1-antiproteinase-like protein

contains only one Cys residue (the other two Cys residues

are located within the putative N-signal sequence). For this

reason, no disulfide bonds are anticipated in the protein

molecule. However, unlike mammals where the Cys residue

is located at position 272, rainbow trout protein has it

(similarly to carp) localized at the N-terminal region (pos.

56 of the deduced cDNA sequence).

Use of the SignalP program [20] allowed for prediction of

the cleavage site for the signal peptide between Ala19 and

Ala20. If this prediction is correct, it implies that additional

cleavage of Ala–Pro dipeptide from protein occurs during

protein maturation, because the N-terminal sequence

obtained after amino acid sequencing of purified protein

starts from His22. Such cleavage might be done by amino-

peptidase or dipeptydyl peptidases. Interestingly, such

enzymes have been identified in human seminal plasma,

including alanyl aminopeptidases and dipeptidyl peptidase

IV that cleaves off N-terminal dipeptides from peptides

containing proline or alanine [42,43]. Aminopeptidase ac-

tivity is also present in fish seminal plasma [44], however,

this activity has not yet been characterized. On the other

hand, although the calculated value of the combined cleav-

age site score [20] is highest for Ala20 (0.869), it is also high

for His22 (0.638). Therefore, it is also possible that the signal

peptide is cleaved between Pro21 and His22 and in such case

the predicted N-terminal sequence will match the sequence

obtained by amino acid sequencing. It seems that carp,

rainbow trout and X. laevis a-APs significantly differ in

the region of the signal peptide. Huang et al. [7] suggested

that a putative junction for the signal peptide and mature

protein is located between Ala16 and Asp17. No signal

peptide has been identified by Yoshida et al. [37] for a1-

antiproteinases of X. laevis.

The physiological role of a1-antiproteinase-like protein

in rainbow trout milt is unknown at present. This inhib-

itor does not appear to be specific for milt because it is

also present in blood and liver, and probably in other

tissues, as was reported for the serpin p62 of carp

perimeningeal fluid [6]. The inhibitor may participate in

protection of spermatozoa from proteolytic attack. Protec-

tion of spermatozoa may be important for successful

prolonged storage of spermatozoa in the spermatic duct

which is characteristic for rainbow trout. Potential target

proteinases for the inhibitor may originate from seminal

plasma, damaged spermatozoa, or blood cells. The pres-

ence of serine proteinases in seminal plasma of rainbow

trout has already been indicated in our laboratory, using

SDS-gelatin-PAGE [45]. Our studies on rainbow trout

seminal plasma also revealed a presence of potential

non-target proteinases for a1-antiproteinase, such as met-

alloproteinases [46,47]. Both blood cells, present in

rainbow trout milt [48], and damaged spermatozoa [49],

are a potential source of serine proteinases. The protective

role of rainbow trout a1-antiproteinase-like protein against

spermatozoa may also include its potential antiapoptotic

and bacteriostatic actions [50,51]. It has to be pointed out

that in addition to protease inhibitory functions, serpins

may influence cell behavior via both direct and indirect

ways. For example, a1-PI may play a role in influencing

tissue repair by directly stimulating fibroblast proliferation

and extracellular matrix production via classical mitogen-

activated signaling pathways [52]. Spermatogenesis con-

sists of many proliferation steps and it is possible that

this process may be controlled by proteinase inhibitors.

The use of pure preparations of a1-antiproteinase-like

protein may be beneficial in further experiments aimed

at the examination of its role in the reproduction of

rainbow trout.

Acknowledgements

This study was supported in part by State Committee for

Scientific Research, Project 5 P06D 027 19 and funds

appropriated to Faculty of Biotechnology, Jagiellonian

Fig. 9. Homology alignment of reactive site sequences of a1-AP of fish, amphibia and mammals. (1) Rainbow trout, (2) carp homolog precursor, (3) carp CP9,

(4) X. leavis, (5) rabbit, and (6) horse. Identical amino acids are marked in bold. Sh = sheet; St = strand, after Ref. [3].


University and Institute of Biochemistry and Molecular

Biology, Wroclaw University. Thanks are due to Michael H.

Penn for editing the manuscript.

References

[1] J. Potempa, E. Korzus, J. Travis, The serpin superfamily of protein

inhibitors: structure, functions, and regulation, J. Biol. Chem. 269

(1994) 15957–15960.

[2] J.A. Irving, R.N. Pike, A.M. Lesk, J.C. Whisstock, Phylogeny of the

serpin superfamily: implications of patterns of amino acid conserva-

tion for structure and function, Genome Res. 10 (2000) 1845–1864.

[3] S.D. Patterson, Mammalian a1-antitrypsins: comparative biochemis-

try and genetics of the major plasma serpin, Comp. Biochem. Physiol.

100B (1991) 439–454.

[4] K. Dabrowski, A. Ciereszko, Proteinase inihibitor(s) in seminal plas-

ma of teleost fish, J. Fish Biol. 45 (1994) 801–809.

[5] A. Ciereszko, B. Piros, K. Dabrowski, D. Kucharczyk,M.J. Luczynski,

S. Dobosz, J. Glogowski, Serine proteinase inhibitors of seminal

plasma of teleost fish: distribution of activity, electrophoretic pro-

files and relation to proteinase inhibitors of blood, J. Fish Biol. 53

(1998) 1292–1305.

[6] C.-J. Huang, C.-C. Chen, H.-.J. Chen, F.-L. Huang, G.D. Chang, A

protease inhibitor of the serpin family is a major protein in carp

perimeningeal fluid: I. Protein purification and characterization, J.

Neurochem. 64 (1995) 1715–1720.

[7] C.-J. Huang, M.-S. Lee, F.-L. Huang, G.-D. Chang, A protease in-

hibitor of the serpin family is a major protein in carp perimeningeal

fluid: II. cDNA cloning, sequence analysis, and Escherichia coli ex-

pression, J. Neurochem. 64 (1995) 1721–1727.

[8] A. Ciereszko, M. Kwasnik, K. Dabrowski, B. Piros, J. Glogowski,

Chromatographic separation of trypsin-inhibitory activity of rainbow

trout blood and seminal plasma, Fish Shellfish Immunol. 10 (2000)

91–94.

[9] M.M. Bradford, A rapid and sensitive method for quantitation of

microgram quantities of protein utilizing the principle of protein

dye-binding, Anal. Biochem. 72 (1976) 248–254.

[10] U.K. Laemmli, Cleavage of structural proteins during the assembly of

the head bacteriophage T4, Nature 227 (1970) 680–685.

[11] J. Uriel, J. Berges, Characterization of natural inhibitors of trypsin and

chymotrypsin by electrophoresis in acrylamide-agarose gels, Nature

218 (1968) 578–580.

[12] R. Geiger, H. Fritz, Trypsin, in: H.U. Bergmeyer (Ed.), Methods

of Enzymatic Analysis, vol. 5. Verlag Chemie, Weinheim, 1983,

pp. 121–124.

[13] K. Olden, K.M. Yamada, Direct detection of antigens in sodium

dodecyl sulfate-polyacrylamide gels, Anal. Biochem. 78 (1977)

483–490.

[14] J.C. Clegg, Glycoprotein detection in nitrocellulose transfers of elec-

trophoretically separated protein mixtures using concanavalin A and

peroxidase: application to arenavirus and flavivirus proteins, Anal.

Biochem. 127 (1982) 389–994.

[15] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins

from polyacrylamide gels to nitrocellulose sheets: procedure and some

applications, Proc. Natl. Acad. Sci. U. S. A. 76 (1979) 4350–4354.

[16] T. Chase Jr., E.S. Show, Titration of trypsin, plasmin and thrombin

with p-nitrophenyl guanidobenzoate, Methods Enzymol. 19 (1970)

20–27.

[17] J. Bieth, Pathophysiological interpretation of kinetic constants of pro-

tease inhibitors, Bull. Eur. Physiopathol. Respir. 16 (1980) 183–195.

[18] H. Schagger, G. von Jagow, Tricine-sodium dodecyl sulfate-poly-

acrylamide gel electrophoresis for the separation of proteins in the

range from 1 to 100 kDa, Anal. Biochem. 166 (1987) 368–379.

[19] L. Falquet, M. Pagni, P. Bucher, N. Hulo, C.J. Sigrist, K. Hofmann,

A. Bairoch, The PROSITE database, its status in 2002, Nucleic Acids

Res. 30 (2002) 235–238.

[20] H. Nielson, J. Englbrecht, S. Brunak, G. von Heijne, Identification of

prokaryotic and eukaryotic signal peptides and prediction of their

cleavage sites, Protein Eng. 10 (1997) 1–6.

[21] F. Aranishi, Purification and characterization of serum serpin from

carp (Cyprinus carpio), Mar. Biotechnol. 1 (1999) 81–88.

[22] F. Aranishi, Purification and characterization of a1-proteinase inhib-

itor from carp (Cyprinus carpio) serum, Mar. Biotechnol. 1 (1999)

33–43.

[23] Ø. Andersen, R. Flengsrud, K. Norberg, R. Salte, Salmon antithrom-

bin has only three carbohydrate side chains, and shows functional

similarities to human h-antithrombin, Eur. J. Biochem. 267 (2000)

1651–1657.

[24] A. Koj, M.W. Hatton, K.L. Wong, E. Regoeczi, Isolation and partial

characterization of rabbit plasma alpha1-antitrypsin, Biochem. J. 169

(1978) 589–596.

[25] Y. Suzuki, K. Yoshida, T. Ichimiya, T. Yamamoto, H. Sinohara, Tryp-

sin inhibitors in guinea pig plasma: isolation and characterization of

contrapsin and two isoforms of a-1-antiproteinase and acute phase

response of four major trypsin inhibitors, J. Biochem. 107 (1990)

173–179.

[26] K. McGilligan, D.W. Thomas, Evaluation of assays for detecting

alpha-1-protease inhibitor during purification from rat serum, Anal.

Biochem. 193 (1991) 260–265.

[27] E. Mattes, H.P. Matthiessen, P.L. Tureck, H.P. Schwarz, Preparation

and properties of an alpha-1-protease inhibitor concentrate with high

specific activity, Vox Sang. 81 (2001) 29–36.

[28] C. Bohme, M. Nimtz, E. Grabenhorst, H.S. Conradt, A. Strathmann,

H. Ragg, Tyrosine sulfation and N-glycosylation of human heparin

cofactor II from plasma and recombinant Chinese hamster ovary cells

and their effects on heparin binding, Eur. J. Biochem. 269 (2002)

977–988.

[29] V.A. Odero-Marah, Z. Khalkhali-Ellis, G.B. Schneider, E.A. Seftor,

R.E.B. Seftor, J.G. Koland, M.J.C. Hendrix, Tyrosine phosphoryla-

tion of maspin in normal mammary epithelia and breast cancer cells,

Biochem. Biophys. Res. Commun. 295 (2002) 800–805.

[30] M.R. Peltier, L.C. Raley, D.A. Liberles, S.A. Benner, P.J. Hansen,

Evolutionary history of the uterine serpins, J. Exp. Zool. 288 (2000)

165–174.

[31] C.Y. Chen, U. Cronshagen, H.F. Kern, A novel pancreas-specific

serpin (ZG-46p) localizes to the soluble and membrane fraction of

the Golgi complex and the zymogen granules of acinar cells, Eur. J.

Cell Biol. 73 (1997) 205–214.

[32] A. Saito, H. Sinohara, Isolation and characterization of rabbit plasma

a-1-antiproteinase E, Biol. Chem. 379 (1998) 1367–1370.

[33] Y.S. Askew, S.C. Pak, C.J. Luke, D.J. Askew, S. Cataltepe, D.R.

Mills, H. Kato, J. Lehoczky, K. Dewar, B. Birren, G.A. Silverman,

SERPINB12 is a novel member of the human ov-serpin family that is

widely expressed and inhibits trypsin-like serine proteinases, J. Biol.

Chem. 276 (2001) 49320–49330.

[34] J. Travis, G.S. Salvesen, Human plasma proteinase inhibitors, Ann.

Rev. Biochem. 52 (1983) 655–709.

[35] Y. Suzuki, K. Yoshida, E. Honda, H. Sinohara, Molecular cloning and

sequence analysis of cDNAs coding for guinea pig alpha 1-antipro-

teinases S and F and contrapsin, J. Biol. Chem. 266 (1991) 928–932.

[36] A. Saito, H. Sinohara, Cloning and sequencing of cDNA coding for

rabbit alpha-1-antiproteinase F: amino acid sequence comparison of

alpha-1-antiproteinases of six mammals, J. Biochem. 109 (1991)

158–162.

[37] K. Yoshida, Y. Suzuki, H. Sinohara, Cloning and comparative se-

quence analysis of Xenopus laevis alpha1-antiproteinase, J. Biochem.

Mol. Biol. Biophys. 3 (1999) 59–63.

[38] N. Takamatsu, M. Kojima, M. Taniyama, K. Ohba, T. Uematsu, C.

Segawa, S. Tsutou, M. Watanabe, J. Kondo, N. Kondo, T. Shiba,

Expression of multiple alpha1-antitrypsin-like genes in hibernating

species of the squirrel family, Gene 204 (1997) 127–132.


[39] J. Haspel, F.W. Sunderman Jr., S.M. Hopfer, D.C. Henjum, P.W.

Brandt-Rauf, I.B. Weinstein, S. Nishimura, Z. Yamaizumi, M.R. Pin-

cus, A nickel-binding serpin, pNiXa, induces maturation of Xenopus

oocytes and shows synergism with oncogenic ras-p21 protein, Res.

Commun. Chem. Pathol. Pharmacol. 79 (1993) 131–140.

[40] F.W. Sunderman Jr., A.H. Varghese, O.S. Kroftova, S. Grbac-

Ivankovic, J. Kotyza, A.K. Datta, M. Davis, W. Bal, K.S. Kasparzak,

Characterization of pNiXa, a serpin of Xenopus laevis oocytes and

embryos, and its histidine-rich Ni(II)-binding domain, Mol. Reprod.

Dev. 44 (1996) 507–524.

[41] B.L. Beck, D.C. Henjum, K. Antonijczuk, O. Zahaia, G. Korza, J.

Ozols, S.M. Hopfer, A.M. Barber, F.W. Sunderman Jr., pNiXa, a

Ni(2+)-binding protein in Xenopus oocytes and embryos, shows iden-

tity to Ep45, an estrogen-regulated hepatic serpin, Res. Commun.

Chem. Pathol. Pharmacol. 77 (1992) 3–16.

[42] I. DeMeester, G. Vanhoof, A.M. Lambeir, S. Scharpe, Use of immo-

bilized adenosine deaminase (EC 3.5.4.4) for the rapid purification of

native human CD26 dipeptidyl peptidase IV (EC 3.4.14.5), J. Immu-

nol. Methods 189 (1996) 99–105.

[43] D. Fernandez, A. Valdivia, J. Irazusta, C. Ochoa, L. Casis, Peptidase

activities in human semen, Peptides 23 (2002) 461–468.

[44] F. Lahnsteiner, R.A. Patzner, T. Weismann, Testicular main ducts and

spermatic ducts in some cyprinid fishes: I. Morphology, fine structure

and histochemistry, J. Fish Biol. 44 (1994) 937–951.

[45] R. Kowalski, J. Glogowski, D. Kucharczyk, K. Goryczko, S. Dobosz,

A. Ciereszko, Proteolytic activity and electrophoretic profiles of

proteases from seminal plasma of teleosts, J. Fish Biol. 63 (2003)

1008–1019.

[46] R. Kowalski, M. Wojtczak, J. Glogowski, A. Ciereszko, Gelatinolytic

and anti-trypsin activities in seminal plasma of common carp: rela-

tionship to blood, skin mucus and spermatozoa, Aquat. Living

Resour. 16 (2003) 438–444.

[47] P.E. Desrochers, K. Mookhtiar, H.E. Van Wart, K.A. Hasty, S.J.

Weiss, Proteolytic inactivation of alpha 1-proteinase inhibitor and

alpha 1-antichymotrypsin by oxidatively activated human neutrophil

metalloproteinases, J. Biol. Chem. 267 (1992) 5005–5012.

[48] T. Wlasow, J. Glogowski, A. Ciereszko, Presence of blood cells in

rainbow trout milt, Pol. Arch. Fish. 7 (1999) 359–364.

[49] K. Inaba, M. Morisawa, Chymotrypsin-like protease activity associ-

ated with demembraned sperm of chum salmon, Biol. Cell 76 (1992)

329–333.

[50] Y. Miyamoto, T. Akaike, H. Maeda, S-nitrosylated human alpha(1)-

protease inhibitor, BBA Prot. Struct. Mol. Enzymol. 1477 (2000)

90–97.

[51] Y. Ikari, E. Mulvihill, S.M. Schwartz, Alpha 1-Proteinase inhibitor,

alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antia-

poptotic factors of vascular smooth muscle cells, J. Biol. Chem. 276

(2001) 11798–11803.

[52] K. Dabbagh, G.J. Laurent, A. Shock, P. Leoni, J. Papakrivopoulou,

R.C. Chambers, Alpha-1-antitrypsin stimulates fibroblast proliferation

and procollagen production and activates classical MAP kinase sig-

nalling pathways, J. Cell. Physiol. 186 (2001) 73–81.


Isolation, characterization, and cDNA sequencing of α-1-antiproteinase-like protein from rainbow trout seminal plasma

Documents