Purification and characterization of novel ... - Wiley Online Library

Eur. J. Biochem. 269, 2639–2646 (2002) FEBS 2002

doi:10.1046/j.1432-1033.2002.02927.x

Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod Anne Ylo¨nen1, Jari Helin1, Jarl Bøgwald2, Anu Jaakola1, Ari Rinne3 and Nisse Kalkkinen1 1

Institute of Biotechnology, Protein Chemistry Laboratory, University of Helsinki, Finland, 2Norwegian College of Fishery Science, University of Tromsø, Norway, 3Institute of Medical Biology, University of Tromsø, Norway

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish (Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDITOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kinin-

Kininogens are large molecular mass (50–114 kDa) cysteine proteinase inhibitors belonging to class 3 of the cystatin superfamily. They are single-chain proteins composed of an N-terminal heavy chain, the bradykinin moiety and a C-terminal light chain. The heavy chain and light chain are interlinked by disulfide bridges. Kininogens are further classified into high molecular mass and low molecular mass kininogens bearing identical heavy chains but the length and the amino-acid sequence of the light chain varies. A vasoactive peptide, bradykinin, is released from kininogens by kallikreins [1]. In addition to carrying bradykinin, kininogens have many other biological functions. They participate in intrinsic blood coagulation and in acute phase reactions as well as inhibit cysteine proteinases [2]. Kininogens have been characterized from many mammals including human, bovine, rat [1], and recently whale [3]. In addition, there are reports on bradykinins in fish including steelhead trout [4] and Atlantic cod [5]. We have recently isolated a kininogen and another high molecular mass cysteine proteinase inhibitor from the skin of Atlantic salmon [6]. To our knowledge, no other kininogen from fish has been described so far. Here, we describe the purification of kininogens from the skin of spotted wolffish (Anarhichas minor) and Atlantic cod (Gadus morhua L.). These novel kininogens were

ogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried a2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units. Keywords: Atlantic cod; spotted wolffish; kininogen; N-glycosylation; O-acetylation.

characterized by determining their molecular mases, partial amino-acid sequences, glycan structures, isoelectric points, as well as their inhibitory activities.

MATERIALS AND METHODS Fish skin samples The starting material for the purifications were the skin of spotted wolffish (Anarhichas minor) and Atlantic cod (Gadus morhua L.) weighing 2–3 kg. The skin (1 kg) was homogenized in 1 L of 10 mM Tris/HCl pH 7.4, 10 mM EDTA, 0.25 M sucrose, 0.1 mM phenylmethanesulfonyl fluoride, 5 mM benzamidine and 15 mM sodium azide. The homogenate was centrifuged at 6000 g for 30 min at 4 C, and the supernatant was collected. To further clarify the supernatant, it was ultracentrifuged at 100 000 g for 2 h at 4 C. The clear extract was collected underneath the floating fat. Purification and characterization of inhibitors The inhibitors from skin extracts were purified as described [6]. After ultracentrifugation the clarified skin extract was subjected to papain-affinity chromatography, gel filtration, anion-exchange chromatography and reversed-phase chromatography. Mass spectrometry

Correspondence to A. Ylo¨nen, Protein Chemistry Laboratory, Institute of Biotechnology, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland. Fax: + 358 9191 59416, Tel.: + 358 9191 59414, E-mail: anne.ylonen@helsinki.fi Enzymes: papain (EC 3.4.22.2); ficin (EC 3.4.22.3); trypsin (EC 3.4.21.4); glutamyl endopeptidase GluC (EC 3.4.21.19). (Received 5 December 2001, revised 2 April 2002, accepted 10 April 2002)

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a BiflexTM time-of-flight instrument (Bruker Daltonik, Bremen, Germany) equipped with a nitrogen laser operating at 337 nm. Proteins were analysed in sinapic acid (Fluka Chemie AG, Buchs, Switzerland), as described previously [6]. Glycans were analysed using 2,4,6-trihydroxyacetophenone (Fluka) or 2,5-dihydroxybenzoic acid (Aldrich,

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2640 A. Ylo¨nen et al. (Eur. J. Biochem. 269)

Steinheim, Germany) as the matrix as described previously [7]. Peptides were analysed using a-cyano-4-hydroxycinnamic acid matrix (Aldrich) as described previously [7]. Capillary isoelectric focusing Capillary isoelectric focusing was performed by using a BioFocus 3000 Capillary Electrophoresis System (BioRad, Richmond, CA, USA). Focusing was performed in Bio-Lyte Ampholyte (pH 3–10, Bio-Rad), in an eCAP neutral capillary (50 lm · 50 cm, Beckman Instruments, Fullerton, CA, USA) according to the manufacturer’s instructions. BioMark CIEF markers (pI values 5.3, 6.4, 7.4, 8.4 and 10.4, Bio-Rad) were used for internal calibration. SDS/PAGE and electroblotting The proteins were separated by SDS/PAGE in a 12% polyacrylamide (w/v) gel [8] and stained with silver [9]. For N-terminal sequencing, the SDS/PAGE separated proteins were subjected to electroblotting on a poly(vinylidene difluoride) membrane (ProBlott, PerkinElmer, Applied Biosystems, CA, USA) in 10 mM 3-(cyclohexylamino)propane-1-sulfonic acid (pH 11)/10% (v/v) methanol with a constant potential of 50 V for 120 min [10]. After staining with Coomassie Brilliant Blue (0.1% in 1% acetic acid/40% methanol) and destaining (50% methanol) the protein bands were cut out and loaded on the sequencer.

Glycan isolation Samples ( 30 lg) of wolffish kininogen and cod kininogen were subjected to enzymatic N-glycan removal with N-glycosidase F. The dry protein sample was dissolved in 100 lL of 20 mM sodium phosphate buffer containing 0.1% SDS and 1% b-octylglucoside, and 2 U of N-glycosidase-F (Roche Biochemicals, Switzerland) were added. After 48 h incubation at 37 C, the reaction mixture was diluted with 4 vol. of water, and the de-N-glycosylated protein was adsorbed to poly(vinylidene difluoride) membrane by centrifugation in a ProSpinTM sample preparation cartridge (PerkinElmer Applied Biosystems Division). The N-glycans were collected from the poly(vinylidene difluoride) flow-through, and were further purified by passing through a BondElut C18 extraction cartridge (Varian SPP, Harbor City, CA, USA) and by gel filtration chromatography as described previously [12]. No separation of the individual N-glycan components was attempted prior to mass spectrometric analyses. The poly(vinylidene difluoride) membrane carrying the de-N-glycosylated protein was subjected to b-elimination to liberate potential O-glycosidically linked glycans. The membrane was incubated in 150 lL of 1 M NaBH4 in 0.1 M NaOH for 48 h at 37 C, and the liberated oligosaccharide alditols were recovered as described previously [12]. O-Glycan fractions were subjected to permethylation [13] prior to MALDI-TOF MS analysis. Removal of O-acetyl groups

Alkylation, enzymatic digestion and peptide separation For in-gel digestion, the proteins were separated by SDS/ PAGE and stained with Coomassie Brilliant Blue (0.1% in 0.5% acetic acid/30% methanol) and destained (30% methanol). The excised protein bands were alkylated with iodoacetamide and digested with trypsin as described previously [11]. For in-solution digestion, the purified proteins from reversed-phase chromatography were alkylated with 4-vinylpyridine and enzymatically digested with trypsin [6] or endoproteinase GluC. For endoproteinase, GluC digestion a 30-lg sample of alkylated protein was dissolved in 50 lL of 50 mM ammonium acetate, pH 4.3 and digested with 0.5 lg of endoproteinase GluC by incubation at 37 C overnight. Generated peptides were separated by reversed-phase chromatography. Sequence analysis Protein N-terminal sequencing and internal peptide sequencing were performed with a Procise 494A sequencer (PerkinElmer Applied Biosystems Division). Inhibition assay After each chromatographic step the inhibitory activity of collected fractions was determined using papain as the enzyme in the assay [6]. The same assay was used to determine inhibitory activites of purified proteins against papain, ficin and trypsin with BANA (Na-benzoyl-DLarginine-2-naphtylamide, Fluka Chemie AG) as a substrate. Positive controls were leupeptin for papain and ficin and phenylmethanesulfonylfluoride for trypsin.

The isolated N-glycans were saponified as described previously [14]. Glycosidase digestions Digestions with Newcastle disease virus (NDV) neuraminidase and Streptococcus pneumoniae b1,4-galactosidase (Oxford Glycosciences, Abingdon, UK) were carried out as described previously [14]. Digestion with jack bean b-galactosidase (Glyko, Novato, CA, USA) was carried out in 10 lL of 100 mM sodium citrate, pH 4.5, at 37 C. An aliquot of 1.5 lL was removed after 16 h of digestion, dropdialysed against water and analysed by MALDI-TOF MS. Phosphatase treatment Dry N-glycan samples were dissolved in 50 mM ammonium bicarbonate, and 1 U of calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA, USA) was added. Synthetic phosphorylated peptides were used as positive controls. After a 2-h incubation at 37 C. the solution was passed through a POROS R3 (PerSeptive Biosystems, Framingham MA, USA) tip, and a sample of the flowthrough carrying the glycans was drop-dialysed and analysed by MALDI-TOF MS.

RESULTS Isolation of cysteine proteinase inhibitors The first step in cysteine proteinase inhibitor isolation from the cod skin extract was affinity chromatography on

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Novel kininogens from cod and wolffish (Eur. J. Biochem. 269) 2641

immobilized papain. Cysteine proteinase inhibitors bound into papain matrix were eluted and the fractions showing over 70% inhibitory activity in the inhibition assay were pooled, concentrated and further purified by gel filtration. The fractions from gel filtration exhibiting inhibitory activity were subjected to anion-exchange chromatography, yielding a single major protein peak expressing inhibitory activity. This fraction was further subjected to reversedphase chromatography and the protein obtained was analysed by SDS/PAGE and MALDI-TOF spectrometry (Fig. 1). In SDS/PAGE, the inhibitor from cod skin migrated at a position corresponding to 78 kDa, whereas MALDI-TOF mass spectrometry gave a molecular mass of 51.0 kDa. Apparently, the glycans on this inhibitor result in the slower migration in SDS/PAGE than expected. Some purified cod inhibitor samples carried also a very faint 60-kDa band as analysed by SDS/PAGE. The relationship between the major 78 kDa and minor 60 kDa gel bands were investigated by in-gel digestion and peptide mass fingerprinting. All peptide signals present in the MALDITOF mass maps from the 60 kDa band were also present in the mass map from the 78-kDa band, suggesting that the 60-kDa band represents a fragment of the 78-kDa band. Similar related forms were detected also in salmon kininogen [6]. In the tryptic mass map, we also detected peptide masses which were tentatively identified as fish bradykinin (m/z 1065.59) peptide as well as fish Arg-bradykinin (m/z 1221.72), previously detected also from salmon kininogen tryptic digest. These results were verified by peptide sequencing [RPPGWSPLR and RRPPGWSPLR (Table 1)]. Accordingly, this cysteine proteinase inhibitor was named cod kininogen. Several isoelectric forms (pI values 3.6, 3.9 and 4.4) of this kininogen were detected by capillary isoelectric focusing, which may be partly ascribed to heterogeneous glycosylation. The yield of reversed-phase purified cod kininogen from 200 mL of cod skin extract was 800 lg.

A cysteine proteinase inhibitor was isolated from wolffish skin extract as above. The wolffish inhibitor purified by reversed-phase chromatography still exhibited two bands in SDS/PAGE, 67 and 42 kDa (Fig. 1). In MALDI-TOF MS, this fraction only shows one clear signal at 45.8 kDa (Fig. 1), probably representing the 67-kDa band that stains poorly in gels. Similar behaviour was observed for salmon kininogen [6], and it is possible that the poor staining may be partly the result of extensive glycosylation. Both bands of the wolffish inhibitor were investigated by in-gel digestion and mass mapping. As above, the peptide patterns obtained were similar, and both included signals appropriate for the fish bradykinin and Arg-bradykinin sequences. Peptide sequencing verified these observations and therefore the cysteine proteinase inhibitor of wolffish skin was named wolffish kininogen. Several forms of the protein with different isoelectric points were again observed (pI values 4.1, 4.3, 4.35, 4.4). The yield of reversed phase purified wolffish kininogen was 850 lg from 200 mL of skin extract. We have previously isolated two high molecular mass cysteine proteinase inhibitors from the skin of Atlantic salmon, a kininogen and a 42-kDa protein named salarin [6]. An identical experimental approach was used in the present study, but interestingly no salarin type inhibitor was detected in the skin of wolffish or cod. Inhibition studies The inhibitory activity of cod and wolffish kininogen was measured with samples purified by reversed phase chromatography. Both kininogens were found to inhibit papain and ficin (cysteine proteinases) but neither of them had any effect on trypsin (a serine proteinase). Their specific activities against papain were calculated from inhibition curves (Fig. 2) and were found to be 940 UÆmg)1 for cod kininogen and 6200 UÆmg)1 for wolffish kininogen (units are defined

Fig. 1. SDS/PAGE visualized by silver staining of purified cod kininogen and wolf fish kininogen, and MALDI-TOF mass spectra of kininogens purified by reversed-phase chramotography.

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Table 1. Peptide sequences determined from cod kininogen (A) and wolffish kininogen (B). X ¼ amino acid not detected. N ¼ glycosylated asparagine according to Edman degradation and mass spectrometry. 5

10

15

20

25

30

A 1 2 3 4 5 6 7 8 9

A E R R I I Q D Y

A F H R C S V S S

V S E P M T V D L

T P V P G G A C Y

S P P G C N G P F

F A Q W P M L A D

N P A S V V Ra G M

E P N P E A

K S L L L L

G R

1 2 3 4 5 6 7 8 9 10 11

X K Q R A S D S Y F F

L A V K G E L D N N P

V P Y K A N N C S E L

Q C C P L G D P M R S

P L L I P S L A S L V

G G L S T D C G D S S

V C D C M S V S S T I

L P D N F V P N T G S

R E Ra,b D Y

A C

E D

E

G

A

M

D

L Q

E I

S L

E S

E A

L S

K V Ka

P

A K

V

T

W

T

Da

I M V A T Y D K H H Ka

F E I T R S D P L Ka

C V I V R L Q W F

D D P Y R Q N T T

D E E M P F A E L

P N K T P T G C H

Sc S

E

D

L

K

E G S Y D F

T W Ra,d A Y V

E S

A P

D L

N L X

C N S Y Ya

L

Ka V

A

V

S

I

S

Ka

V

A

d

B

a

Peptide aligned with human kininogen.

b

T Kd Ra,b S E

T R

Contains fish bradykinin sequence. c N-Terminus of fragment.

V N Ra

d

Glycopeptide.

Determination of partial amino-acid sequences

Fig. 2. Inhibition of papain (0.625 mg) by different amounts of cod kininogen and wolffish kininogen.

as lg of papain inhibited per mg of inhibitor, data not shown). The inhibitory activity of cod kininogen and wolffish kininogen has also been investigated with different cathepsins isolated from cod, wolffish and salmon (E. Weber, Institute of Physiological Chemistry, MartinLuther-University, Halle-Wittenberg, Germany, personal communication). These experiments suggest that these kininogens inhibit specifically cathepsin L but not cathepsin B or cathepsin H.

N-Terminal sequencing of the kininogens was only successful from the 42-kDa band of wolffish kininogen (XLVQPGVLI…, Table 1). The major bands of both kininogens and also the 60-kDa band of cod kininogen were found to be N-terminally blocked. Peptides were generated from both kininogens by digesting the alkylated proteins with trypsin or endoproteinase GluC. The peptides were separated by reversed phase chromatography and selected peptides were subjected to sequence analysis. A few overlapping sequences from trypsin and endoproteinase GluC digested peptides were obtained and the results from sequencing are shown in Tables 1. Both cod and wolffish kininogens carried the fish bradykinin peptide RPPGWSPLR that we have previously shown to be present in Atlantic salmon kininogen [6]. The fish bradykinin peptide has also been found previously from enzyme-treated plasma of Atlantic cod [5] and steelhead trout [4]. Most of the determined peptide sequences could be aligned with the heavy chain of human kininogen (Fig. 3), verifying the identity of these novel kininogens. The highly conserved cystatin sequence QVVAG was also found among the cod sequences. The complete primary structures of cod kininogen and wolffish kininogen will be revealed by molecular cloning, which also enables more detailed homology and phylogenetic comparisons. Glycosylation studies The difference between the apparent Mr in SDS/PAGE and that analysed by MALDI-TOF MS implied that both kininogens are glycosylated. To release potential N-glycans

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Fig. 3. Alignment of peptides from cod kininogen (COD KIN) and wolf fish kininogen (WF KIN) with heavy chain of human kininogen (KNH-HUM, P01042) sequence. Identical amino acids are shaded.

for structural characterization, samples of the kininogens were subjected to N-glycan liberation by N-glycosidase F. The released glycans were isolated by gel filtration and N-glycan pools were analysed by MALDI-TOF MS. The spectrum of cod kininogen N-glycans is shown in Fig. 4A, revealing an extremely heterogeneous pattern. Two major clusters of ions are observed, and within both clusters adducts of +42 or +80 Da can be revealed. We have recently identified from salmon kininogen N-glycans very high levels of sialic acid O-acetylation [14], and it is probable that the +42 Da adducts in the cod kininogen N-glycans represent this modification as well. This is supported by the higher number of acetyl (Ac) groups in those glycans that carry more sialic acid residues. The +80 Da adduct could be either phosphorylation or sulfation. As will be described below, the ions carrying the +80 Da adduct were stable against alkaline phosphatase treatment, establishing the presence of sulfate (SO3) groups. The assignments are therefore as follows (all [M-H]– ions): m/z 1932.3 and 1973.6, monosialylated biantennary glycans with 0 and 1 acetyl groups, respectively. The signal at m/z 2012.8 is assigned as a monosialylated biantennary glycan with one sulfate (SO3), while the ion at m/z 2053.5 carries one acetyl and one sulfate group. Disialylated biantennary glycans are found at m/z 2222.8, 2264.8 (+1 Ac), 2306.8 (+2 Ac), 2344.9 (+1 Ac, +1 SO3), and 2387.2 (+2 Ac, +1 SO3). Disialylated triantennary species are found at m/z 2588.5, 2630.4 (+1 Ac), 2672.2 (+ 2 Ac), 2710.8 (+ 1 Ac, +1 SO3), 2752.9 (+ 2 Ac, +1 SO3), and finally, trisialylated biantennary glycans at m/z 2879.3, 2921.4 (+1 Ac), 2963.4 (+2 Ac), 3005.4 (+3 Ac), 3044.1 (+2 Ac, +1 SO3) and 3086.1 (+3 Ac, +1 SO3). Short alkaline hydrolysis (saponification) of the N-glycans yielded glycans devoid of additional acetyl groups, an indication that these were O-acetyl substituents [15]. The MALDI-TOF spectrum of the de-O-acetylated glycans reveals the sulfated species very clearly at m/z 2302.8 and m/z 2960.0, representing the monosulfated disialylated biantennary and trisialylated triantennary species, respect-

ively (Fig. 4B). Sulfated species are observed also for the monosialylated biantennary and disialylated triantennary species, at m/z 2012.2 and m/z 2667.6, respectively. To obtain additional data on the glycan structures alkaline phosphatase and exoglycosidase digestions were performed for the de-O-acetylated species. First, the desialylated glycans were treated with alkaline phosphatase. No removal of the +80 Da adducts could be observed under conditions where full dephosphorylation of both phosphorylated glycans and peptides is consistently obtained, thus verifying the adduct’s nature as sulfation (not shown). The glycans were then subjected to Newcastle disease virus (NDV) sialidase treatment, which liberates a2,3-linked but not a2,6-linked sialic acids, and practically complete desialylation was observed (Fig. 4C). The small signal at m/ z 2394.0 may represent desialylated tetraantennary glycans. The sulfate group remained in the oligosaccharide part, although at a reduced intensity. It should be noted that the sulfated glycans are now observed as +102 Da adducts, exhibiting [M-H + 2Na]+ ions. The reduced signal of the sulfated species is probably due to fragmentation in the MALDI-TOF MS analysis, where partial sulfate loss (but not loss of phosphate) is in our experience invariably observed in the positive ion reflector mode. The more gentle negative ion mode cannot be used here, as neutral carbohydrates do not ionize in the negative ion mode. The desialylated glycans were further subjected to digestion with S. pneumoniae b-galactosidase. Under the conditions used, this enzyme liberates b1,4-linked galactose units only. The MALDI-TOF spectrum of the digested glycans shows a complete removal of galactose units from the nonsulfated biantennary (m/z 1663.7 to m/z 1339.5) and triantennary (m/z 2028.9 to m/z 1542.7) species (Fig. 4D). One galactose unit in the sulfated glycans was resistant to the enzyme, as revealed by the signals at m/z 1603.6 (biantennary) and m/z 1806.8 (triantennary). This is as expected, as S. pneumoniae b-galactosidase is not able to hydrolyze galactose from Galb1–4GlcNAc-R type structures where either Gal or GlcNAc is sulfated [16]. However,


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Fig. 4. MALDI-TOF spectra of kininogen N-glycans. (A) Intact cod kininogen N-glycans, and the cod kininogen N-glycans after successive incubations (B) in 0.1 M NaOH (saponification) (C) with NDV sialidase (D) with S. pneumoniae b-galactosidase and (E) jack bean b-galactosidase. (F) Intact wolffish N-glycans, and after successive incubations (G) in 0.1 M NaOH (H) with NDV sialidase and (I) with S. pneumoniae b-galactosidase.The proposed glycan structures are shown: j, GlcNAc; s, mannose; h, galactose; r, N-acetylneuraminic acid; circled S, sulfate. The spectra shown in A, B, F and G were recorded in linear negative ion mode, signals are [M-H]– ions, and average mass values are shown. The spectra of C, D, E, H, and I were obtained in reflector positive ion mode, signals are [M + Na]+ ions with monoisotopic mass values.

b-galactosidase from jack beans is inhibited by sulfate on the Gal unit only, and as shown in Fig. 4E, even the last galactose unit is removed by the latter enzyme (m/z 1603.6 to m/z 1441.4 and m/z 1806.8 to m/z 1644.5). This indicates that the sulfate is linked to the GlcNAc residue. Altogether, the cod kininogen carries biantennary and triantennary N-glycans that are terminated by a2,3-linked sialic acids, a high number of which are O-acetylated. About 1/3 of the glycans carry sulfate at N-acetylglucosamine units of their Neu5Aca2,3Galb1,4GlcNAc antennae.

The isolated N-glycans of the wolffish kininogen exhibited a complicated pattern in MALDI-TOF MS (Fig. 4F), with two major clusters of ions. Adducts of +42 Da are again evident in the spectrum, implying extensive O-acetylation of sialic acids. The signals were tentatively identified as follows (all [M-H]– ions): monosialylated biantennary glycans, m/z 1932.2 and 1974.3 (+1 Ac). Disialylated biantennary, m/z 2223.1, 2265.1 (+1 Ac), 2307.0 (+2 Ac), 2349.3 (+3 Ac) and 2391.2 (+4 Ac). Disialylated triantennary, m/z 2588.7, 2630.5 (+1 Ac),

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2673.1 (+2 Ac), 2714.8 (+3 Ac). Finally, trisialylated triantennary, m/z 2879.8, 2921.5 (+1 Ac), 2963.8 (+2 Ac), 3005.6 (+3 Ac), 3047.7 (+4 Ac), 3089.7 (+5 Ac), 3131.3 (+6 Ac), 3173.7 (+7 Ac). No signals corresponding to sulfated glycans were observed in these N-glycans. The MALDI-TOF spectrum of the saponified wolffish N-glycans was much simpler, exhibiting signals assignable to sialylated biantennary and triantennary glycans (Fig. 4G). Treatment of the de-O-acetylated glycans with NDV sialidase resulted in complete removal of the sialic acid residues (Fig. 4H), indicating that the sialic acids were a2,3-linked. Digestion of the desialylated glycans with S. pneumoniae b-galactosidase abolished all terminal galactose residues (Fig. 4I), showing that these were b1,4-linked. To summarize, wolffish kininogen carried a2,3-sialylated biantennary and triantennary N-glycans, with extensive sialic acid O-acetylation. Also O-glycosidic glycans were recovered from wolffish kininogen. The permethylated O-glycan pool was analysed by MALDI-TOF MS, and the spectrum revealed one major [M + Na]+ signal at m/z 1256.63 (not shown). The mass is appropriate for the composition (Neu5Ac)2(Hex)1(HexNAc-ol)1, suggesting that wolffish kininogen carries disialylated core type-1 O-glycans similar to those previously identified from salmon kininogen [14]. The exact natures of the O-acetylated and sulfated residues is beyond the scope of the present study. Preliminary LC-MS/MS analysis of wolffish kininogen sialic acids as 1,2-diamino-4,5-methylenedioxybenzene derivatives revealed 7-, 8-, and 9-O-acetylation, but no 4-O-acetylation of Neu5Ac. This suggests a similar type of O-acetylation as that recently found in Atlantic salmon glycoproteins [14].

DISCUSSION The present study describes the identification of two novel kininogens from Atlantic cod (Gadus morhua L.) and spotted wolffish (Anarhichas minor). The characteristic bradykinin sequence RPPGWSPLR was demonstrated in both cod and wolffish kininogens. In addition, the highly conserved cystatin peptide QVVAG, which is found in domains 2 and 3 for mammalian kininogens [1], was present in cod kininogen. We have previously characterized a kininogen from Atlantic salmon (Salmo salar L.) as well [6]. In both of these studies, we have used a method that catches large molecular cysteine proteinase inhibitors with papainaffinity chromatography. Kininogens have many biochemically defined properties; they are substrates for kallikreins and release vasoactive peptide bradykinin. Furthermore, they act like cystatins inhibiting cysteine proteinases. Cod and wolffish kininogens and previously isolated salmon kininogen all possess these properties. Kininogen seems to be very conserved both in structure and its effects. We recently characterized the glycosylation of kininogen isolated from Atlantic salmon (Salmo salar L.), which was shown to carry extensively O-acetylated sialic acids as terminal elements on biantennary N-glycans [14]. This structural feature is conserved in the kininogens of cod and spotted wolffish, as shown in the present study. In addition, sulfated N-acetylglucosamine residues were observed in the antennae of cod kininogen N-glycans, but not in kininogens from salmon or spotted wolffish. The biological relevance of

O-acetylation or sulfation in the kininogens remains to be established. It is known that, in general, sialic acid O-acetylation protects glycans from enzymatic degradation, as many sialidases are inhibited by this modification [17]. It is noticeable that Atlantic salmon also carried another high molecular mass (42.7 kDa) cysteine proteinase inhibitor, named salarin [6]. The present study shows no evidence for the existence of this type of protein in Atlantic cod or spotted wolffish. Recently, we isolated from another salmonid species, Arctic charr (Salvelinus alpinus), a 42-kDa papain-inhibiting protein with an N-terminus identical with that of salarin. It is therefore possible that salarin type proteins are typical only to salmonids, and the more evolved fish species analysed in the present study have lost this protein in evolution. Alternatively, salarin-type proteins may have lost their cysteine proteinase inhibitory activity and thus cannot be isolated by papain affinity chromatography.

ACKNOWLEDGEMENTS We wish to thank Dr Mikko Ja¨rvinen for critical review of this manuscript. The study has been carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT97-3508, Fish cysteine proteinase inhibitors and infectious diseases. It does not necessarily reflect its views and in no way anticipates the Commision’s future policy in this area. This study was also partly supported by a grant from the Academy of Finland (grant no. 46692 to J. H.).

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