Glycosylation analysis of two cysteine proteinase inhibitors from Atlantic salmon skin: di-O-acetylated sialic acids are the major sialic acid species on N-glycans

Anne Ylönen2, Nisse Kalkkinen2, Juhani Saarinen2, Jarl Bøgwald3 and Jari Helin1,2

2Institute of Biotechnology, Protein Chemistry Laboratory, P.O. Box 56 (Viikinkaari 9), University of Helsinki, FIN-00014 University of Helsinki, Finland and 3Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway

Received on September 6, 2000; revised on January 5, 2001; accepted on January 29, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We have recently identified two novel cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.), named salmon kininogen and salarin. In preliminary experiments, the proteins were found to be both N- as well as O-glycosylated. In the present study we show that both proteins carry biantennary {alpha}2,3-sialylated N-glycans. A very high amount of O-acetylated Neu5Ac units are present in the N-glycans, comprising about 60% di-O-acetylated species. Non-O-acetylated Neu5Ac make up less than 5% of the sialic acids in the N-glycans. A small number of Neu5Ac{alpha}2-8Neu5Ac structures were observed in the N-glycans as well. O-glycans from both proteins were recovered by reductive beta-elimination and were identified by mass spectrometric methods as mono- and disialylated core type 1 tri- and tetrasaccharides. The method used for O-glycan isolation prevented the identification of possible O-acetylation in the O-glycan-bound sialic acids, but O-acetylation was observed in one O-glycosylated peptide isolated from trypsin digest of salarin. The chemical nature of the sialic acid modifications was further studied by liquid chromatography tandem mass spectrometry of 1,2-diamino-4,5-methylenedioxybenzene–derivatized sialic acids, revealing 7-, 8-, and 9- but no 4-O-acetylation. To our knowledge, these are the first observations of sialic acid O-acetylation in N-glycans on fish species and represent clearly the most extensive N-glycan O-acetylation described on any species.

Key words: cysteine proteinase inhibitor/glycoprotein/mass spectrometry/O-acetylation/sialic acid


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are studying cysteine proteinase inhibitors present in the skin of various northern Atlantic fish species. During the characterization of two novel inhibitors from Atlantic salmon (Salmo salar L.), we found these to be glycoproteins carrying both N- and O-glycosidic glycans (Ylönen et al., 1999Go). The present study was initiated to characterize the detailed structures of both types of glycans in these glycoproteins, as studies on Atlantic salmon glycoproteins are very scarce. Antithrombin from Atlantic salmon has been shown by lectin blotting to carry complex-type, {alpha}2,3-sialylated N-glycans, and no O-glycans were observed (Andersen et al., 2000Go). The closest relative of Atlantic salmon, brown trout (Salmo trutta), has been reported to express glycosylated albumin with sialylated (but otherwise undefined) N-glycans (Metcalf et al., 1998Go).

The glycosylation in other salmonids shows some intriguing features. Very large N-glycans carrying Lewis X and {alpha}2,8-disialyl groups have been described from a rainbow trout (Oncorhynchus mykiss) ovarian fluid glycoprotein (Funakoshi et al., 1997Go). Inoue and Iwasaki described a polysialoglycoprotein from the unfertilized eggs of rainbow trout carrying O-glycosidic oligo-N-glycolylneuraminyl (poly-Neu5Gc) chains (Inoue and Iwasaki, 1978Go). Later studies demonstrated similar structures in other salmonids as well (Iwasaki and Inoue, 1985Go). Of special interest are the poly-Neu5Gc chains of kokanee salmon (Oncorhynchus nerka adonis), in which the Neu5Gc units are O-acetylated at various positions (Iwasaki et al., 1990Go).

O-acetylation is perhaps the most common modification found on sialic acids. These substitutions are widespread in higher animals and are also found in several fungi and many, usually pathogenic, bacteria (Varki, 1992Go; Klein and Roussel, 1998Go; Alviano et al., 1999Go). Some intriguing aspects of the biological significance of sialic acid O-acetylation in vertebrates have been established. The lifetimes of serum glycoproteins and blood cells are extended by O-acetylation of their sialic acid residues, because this substitution confers resistance to the action of most sialidases (Corfield et al., 1986Go), as well as to acid-catalyzed hydrolysis (Varki and Diaz, 1984Go; Vandamme-Feldhaus and Schauer, 1998Go). These substitutions have also been implicated in protection against microorganisms, as, for example, the binding of influenza A and B viruses as well as malarial parasites to target cells is abrogated by sialic acid O-acetylation (for review, see Varki, 1992Go).

In the present study we describe the structures of both N- and O-glycosidic glycans from two cysteine proteinase inhibitors, salmon kininogen and salarin, isolated from Atlantic salmon skin. We present data showing that the N-glycans of these proteins are very highly O-acetylated at their nonreducing terminal sialic acid units, with practically no unsubstituted Neu5Ac present. The major N-glycoproteins in salmon skin, transferrin and GP55, were found to carry similar modifications.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
N-glycosylation of salmon kininogen and salarin
The presence of glycosylation in the two novel cysteine proteinase inhibitors of salmon skin was implied by the difference of the apparent molecular weight (MW) on SDS–polyacrylamide gel electrophoresis (PAGE) and the MW obtained by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Moreover, by treatment with peptide:N-glycosidase F (PNGase F) the apparent MW of both salmon kininogen and salarin was reduced, thus confirming the presence of N-glycosylation (Ylönen et al., 1999Go).

To obtain N-glycans for more detailed structural characterization, a sample of salmon kininogen (30 µg, about 0.6 nmol) was subjected to N-glycan liberation by PNGase F. Isolation of the liberated N-glycans by gel filtration yielded about 1 nmol of oligosaccharides (as determined by UV absorbance), thus implying two N-glycosylation sites. The N-glycan pool was subjected to MALDI-TOF MS (Figure 1A). The spectrum shows three clusters of ions, and within each cluster the ions are observed at 42-Da intervals. This implies variable degree of acetylation in the glycan structures, O-acetylation of sialic acid residues being the most probable alternative (Schauer, 1991Go). The signals were tentatively identified as sialylated biantennary complex-type glycans by their MW: m/z 1932.8, 1974.2, and 2016.4, monosialylated glycans with 0, 1, and 2 acetyl (Ac) groups, respectively; m/z 2222.4, 2264.9 (+ 1 Ac), 2307.1 (+ 2 Ac), 2349.1 (+ 3 Ac), and 2391.1 (+ 4 Ac), disialylated glycans; and m/z 2598.7 (+ 2 Ac), 2640.7 (+ 3 Ac), and 2682.6 (+ 4 Ac), trisialylated glycans (all [M–H] ions). The relative intensities of the signals can be used to estimate the molar ratio of the components in the glycan mixture (Papac et al., 1996Go): the data thus implies that about 60% of sialic acids are di-O-acetylated.



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Fig. 1. MALDI-TOF spectra of salmon kininogen N-glycans. (A) Intact glycans, (B) saponified N-glycans, and (C) glycans after treatment with NDV sialidase. Spectra in (A) and (B) were measured in negative ion mode and (C) in positive ion mode. The signals at m/z 2459.1 and 2481.3 in (B) as well as at m/z 1877.4 and 1899.4 in (C) are matrix adducts typical to trihydroxyacetophenone matrix when samples contain Na-salts.

 
Short alkaline hydrolysis (saponification) of the N-glycans in 0.1 M NaOH yielded glycans devoid of additional acetyl groups, an indication that these indeed were O-acetyl substituents (Varki and Diaz, 1984Go) (Figure 1B). The MALDI-TOF spectrum of the de-O-acetylated glycans reveals a small signal of monosialylated biantennary glycan (m/z 1931.6 [M–H]), disialylated counterparts (m/z 2223.1 [M–H], m/z 2245.2 [M+Na–2H]), and trisialylated counterparts (m/z 2514.3 [M–H], 2536.5 [M+Na–2H], 2558.7 [M+2Na–3H]).

Exoglycosidase digestions followed by MALDI-TOF MS analysis were carried out to obtain structural information of the N-glycans. The intact glycan pool was first treated with Newcastle disease virus (NDV) neuraminidase, which partly removed monoacetylated sialic acids from the glycans (not shown). This data agree with earlier studies on the specificity of neuraminidases against O-acetylated sialoglycoconjugates (Corfield et al., 1986Go). When the glycan pool was de-O-acetylated and treated with NDV neuraminidase (known to remove only {alpha}2,3- and {alpha}2,8-linked sialic acids), a complete removal of sialic acid units was seen (Figure 1C): The MALDI-TOF spectrum now shows one glycan species only, m/z 1663.5 [M+Na]+,1679.5 [M+K]+, with MW appropriate for nonsialylated biantennary N-glycan.

Finally, the desialylated glycans were treated with Streptococcus pneumoniae ß-galactosidase. Under the experimental conditions used, this enzyme is specific for the ß1,4-linkage and no Galß1,3-linkages will be cleaved. Two galactose units were removed as analyzed by MALDI-TOF MS (not shown), a result compatible with ß1,4-galactosylated biantennary structure. To sum up, these results indicate that the N-glycans of salmon kininogen were biantennary glycans terminated by O-acetylated {alpha}2,3-linked sialic acids.

The trisialylated biantennary species carries a Neu5Ac-Neu5Ac disialosyl group at the nonreducing termini of the N-glycan. This was verified by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis of the trisialylated species in permethylated form (not shown). The disialosyl group was {alpha}2,8-linked as established by periodate oxidation of the saponified N-glycans: MALDI-TOF MS analysis of the periodate-oxidized –NaBH4 reduced N-glycans revealed three signals, m/z 1873.6, m/z 2105.2, and m/z 2396.6 ([M–H] ions) (not shown). The m/z 1873.6 and m/z 2105.2 signals are generated from mono- and disialylated glycans, respectively, by the oxidative cleavage of the sialic acid side chain: Each sialic acid is truncated to C7-analogue, and –60 Da loss per each sialic acid is evident in the spectrum. The trisialylated glycan produced m/z 2396.6, indicating that only terminal sialic acids were oxidatively cleaved. This is only possible if the sialic acids are {alpha}2,8-linked, which renders the side chain of the inner sialic acid unit stable against periodate cleavage. This data is supported by the disappearance of the trisialylated glycan signal after treatment with NDV sialidase (Figure 1B,C). It is noteworthy that there was no more than four O-acetyl groups in the trisialylated biantennary glycan (see Figure 1A). This may suggest that only the terminal Neu5Ac unit is an acceptor for the O-acetyltransferase(s).

A sample of 30 µg (approximately 0.7 nmol) of salarin was treated with PNGase F, and 0.5 nmol of N-glycans were isolated by gel filtration, thus implying one N-glycosylation site in this protein. All structural studies described above for salmon kininogen N-glycans were also performed for salarin N-glycans. Practically identical results were obtained (not shown).

Identification of salmon kininogen and salarin N-glycosylation sites
Because the complete amino acid sequence of salmon kininogen or salarin is not known, we attempted to establish the N-glycosylation sites by characterizing glycopeptides from both proteins. Samples of salmon kininogen and salarin (about 30 µg each) were alkylated with 4-vinylpyridine and digested with trypsin. The tryptic peptides were separated by narrow-bore reversed phase (RP) chromatography (Figures 2 and 3), and aliquots of the fractions were subjected to MALDI-TOF MS and N-terminal sequencing. Glycopeptides in our experience seldom produce high-quality MALDI-TOF spectra, and identifying them in, for example, spectra of unseparated tryptic peptide mixtures may be impossible. This may partly be ascribed to their ionization properties, but logically also the heterogeneity of the glycan unit (i.e., glycoforms) will spread the signal over a number of mass values. The latter however offers an immediate possibility to identify high-performance liquid chromatography (HPLC) fractions containing glycopeptides, where, for example, differences of 291 Da (N-acetylneuraminic acid) or 146 Da (fucose) may be observed. For salmon kininogen and salarin, the utmost heterogeneity in the N-glycopeptides is sialic acid acetylation, yielding signals 42 Da apart. This is clearly seen in the spectra of salmon kininogen N-glycopeptides (SK-GPs, Figure 2B,C) and salarin N-glycopeptide (Sr-GP2, Figure 3C). N-terminal sequencing of SK-GP1 yielded AQXDSCTQYFVEFNSR, X representing a probable glycosylated asparagine residue in a N-glycosylation consensus sequence. The signals in the MALDI-TOF spectrum of SK-GP1 are assigned as follows (Figure 2B, Table I): m/z 3639.9, peptide + nonsialylated biantennary glycan; m/z 3930.4, 3972.8 (+ 1 Ac), 4014.9 (+ 2 Ac) monosialylated (O-acetylated) counterpart; m/z 4222.4, 4262.6 (+ 1 Ac), 4304.2 (+ 2 Ac), 4346.1 (+ 3 Ac), 4387.9 (+ 4 Ac), disialylated (O-acetylated) counterparts. Compared to the isolated N-glycans, the glycan part in the glycopeptide shows remarkable differences: The appearance of signals corresponding to nonsialylated and monosialylated structures is probably mostly due to fragmentation in the MALDI-TOF MS analysis (Juhasz and Costello, 1992Go). Furthermore, partial de-O-acetylation is very likely under the conditions trypsin digestion was carried out, accounting for the lower overall O-acetylation in the glycopeptide. SK-GP1 was also treated with PNGase F, and MALDI-TOF MS analysis of the deglycosylated peptide yielded m/z 2014.7 (not shown), a value pertinent for the sequence obtained (considering N->D conversion of the glycosylated residue in the PNGase F treatment).



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Fig. 2. (A) Separation of tryptic peptides from salmon kininogen with narrow-bore RP chromatography. (B) MALDI-TOF spectrum of SK-GP1. (C) MALDI-TOF spectrum of SK-GP2. Both spectra were recorded in positive ion mode with external calibration. See Table I for assignments.

 


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Fig. 3. (A) Separation of tryptic peptides from salarin with narrow-bore RP chromatography. (B) MALDI-TOF spectrum of Sr-GP1, measured in positive ion mode. (C) MALDI-TOF spectrum of Sr-GP2, measured in negative ion mode. See Table I for assignments. Large signals marked with asterisks in (B) are contaminating peptides in the HPLC fraction.

 

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Table I. The signal assignments of glycopeptide MALDI-TOF spectraa
 
The SK-GP2 gave the sequence ELNDECHPDPDVELAHCXSTVDVAPWR, X being the glycosylated residue, and its MALDI-TOF spectrum reveals the expected heterogeneity due to the glycan part (Figure 2C, signal assignments in Table I). Deglycosylation of SK-GP2 generated a peptide with m/z 3272.3 (not shown), appropriate for the sequence.

Two fractions exhibiting similar O-acetylation and sialic acid heterogeneity were observed among the salarin tryptic peptides (Figure 3). Sr-GP1 was, however, found to be stable against PNGase F treatment, thus implying this fraction to be O-glycopeptide, and its structural characterization will be described below. Sr-GP2 yielded sequence AEXGSESFTMGINYFSDMTFEEIPK by N-terminal sequencing, and after de-N-glycosylation the peptide exhibited m/z 2845.1 in MALDI-TOF MS (not shown), confirming that the third amino acid is a glycosylated asparagine. Assignments of the signals in Sr-GP2 MALDI-TOF spectrum (Figure 3C) are shown in Table I.

Glycopeptides derived from both salmon kininogen and salarin elute in HPLC as partially resolved molecular species explaining their appearance in the chromatogram. The more highly O-acetylated species were found to elute later (not shown), as expected by their increased hydrophobicity.

Characterization of O-glycosylation in salmon kininogen and salarin
O-glycosidically bound glycans in salmon kininogen and salarin were liberated by alkaline reductive ß-elimination. The isolated O-glycans were permethylated and analyzed by MALDI-TOF MS. Both proteins were found to carry mostly (Neu5Ac)2(Hex)1(HexNAc-ol)1 species, as deduced from the mass spectrum (not shown). Many salmonids carry oligo–sialic acid structures in polysialoglycoprotein O-glycans (Iwasaki and Inoue, 1985Go), and therefore it was interesting to subject the salmon skin glycoprotein O-glycans to MS/MS, which is a powerful technique in analyzing isomeric glycans (Reinhold et al., 1995Go). Figure 4 shows the MS/MS spectrum of salmon kininogen–derived disialylated O-glycan in permethylated form (parent ion [M+Na]+, m/z 1256.5). Of particular interest in distinguishing between isomeric structures are the following fragment ions: The Y2a/Y1b ion at m/z 506.2 represents a Hex-HexNAc-ol ion, which has lost two substituents, that is, the two sialic acid residues did not form a diNeu5Ac unit. The C2a ion at m/z 620.2 is a Neu5Ac-Gal ion, fragmented most probably from the 3-OH of the reduced GalNAc-ol. The Neu5Ac-GalNAc-ol Y1a-H2O fragment at m/z 659.2 also supports the view that Neu5Ac-Gal was linked to 3-OH of GalNAc-ol, because the loss of 3-substituent is often accompanied by water elimination (Egge and Peter-Katalinic, 1987Go). Taken together, the MS/MS data implies that salmon kininogen tetrasaccharide O-glycan is a core type 1 glycan (Hounsell et al., 1996Go), carrying sialic acids at both Gal and GalNAc-ol units. There was no signal observed at m/z 759, where the B-ion of Neu5Ac-Neu5Ac disialyl group would reside, suggesting that not even a small amount of these structures were present.



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Fig. 4. MS/MS spectrum of permethylated O-glycan from salmon kininogen. The proposed structure with fragment assignments (nomenclature of Damon and Costello, 1988) is also shown.

 
Salarin-derived disialylated O-glycan gave practically identical results in MS/MS (not shown). The monosialylated O-glycans observed in the MALDI-TOF MS in low amounts were not studied further.

Alkaline ß-elimination used in O-glycan isolation destroys O-acetyl substituents, and therefore no evidence was obtained about the presence of O-acetylation in sialic acids of O-glycans. However, as stated above, we found one probable O-glycopeptide among the tryptic peptides of salarin, Sr-GP1 (Figure 3A). This fraction yielded amino acid sequence LMVVFPXRDGGEEAEVDKEFETWK; the nonidentified amino acid is not in an N-glycosylation consensus sequence, and the fraction was resistant to PNGase F, excluding N-glycosylation. The tentative assignment of Sr-GP1 MALDI-TOF spectrum (Figure 3B, Table I) suggested the unidentified amino acid to be threonine, which was verified by gas-phase ß-elimination of the O-glycopeptide in methylamine (Mirgorodskaya and Roepstorff, 1998Go). In this procedure, the O-glycosylated residue loses the glycan part and is converted to a stable methylamine derivative. The mass spectrum of methylamine treated O-glycopeptide revealed a major signal at m/z 2825.2 (not shown), appropriate for the ß-eliminated glycopeptide carrying a methylamine derivative of threonine.

The MALDI-TOF spectrum of Sr-GP1 clearly shows the presence of additional acetylation, implying O-acetylation in the O-glycans of salarin as well. We were not able to detect any O-glycopeptides among the tryptic peptides of salmon kininogen. The O-glycans of this glycoprotein may all reside clustered in one area of the protein backbone that contains no trypsin cleavage sites.

Characterization of sialic acid species
Sialic acids from salmon kininogen and salarin were isolated by hydrolysis in 2 M propionic acid (Mawhinney and Chance, 1994Go), and purified from proteins by running the hydrolysate through a POROS R1 micro-column. These hydrolysis conditions have been developed to minimize the loss of O-acetyl substituents. The corresponding sialic acid quinoxalinone derivatives were formed by reaction with 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent (Hara et al., 1989Go). Preliminary analysis of the sialic acid–DMB derivatives from both salarin and salmon kininogen by LC-MS indicated that Neu5Ac and its mono- and di-O-acetylated species are the only sialic acids present (not shown). Identification of these components by LC-MS/MS is shown in Figure 5. The eluting peaks were identified by MW and characteristic MS/MS fragment pattern (data not shown) as well as by their elution order according to Klein et al. (1997)Go, and the proposed structures are indicated in Figure 5. It is probable that the quinoxalinone part largely dictates the ionization properties of the derivatives (all [M+H]+ ions), and thus the peak intensities may be used for estimating the molar ratio of the sialic acid species. The LC-MS/MS analysis showed clearly that 4-O-acetylation is absent in these glycoproteins. The major mono-O-acetylated sialic acid species was identified as Neu5,9Ac2. Neu5,7Ac2 and Neu5,8Ac2 were present as well. The di-O-acetylated species expectedly comprise the three possible structures, carrying O-acetyl groups in 7-, 8-, and 9-OH of Neu5Ac. However, at this stage we are not able to identify the di-O-acetylated isomers in the chromatogram. Attempts to assign the MS/MS spectra of the peaks have so far been unsuccessful. Neither have we found studies in the literature where the relative elution order of di-O-acetylated sialic acid DMB-derivatives would have been reported.



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Fig. 5. Characterization of sialic acid species by LC-MS/MS. Sialic acids were liberated from proteins by acid hydrolysis and derivatized with DMB. DMB–sialic acid derivatives were separated on microbore RP-HPLC and detected by Q-TOF mass spectrometer as described in the text.

 
The relatively high amount of non-O-acetylated Neu5Ac observed in LC-MS may indicate that O-glycans are not as highly O-acetylated as N-glycans. Indeed, the disialylated species of salarin O-glycopeptide Sr-GP1 carries no more than two O-acetyl groups, in contrast to the disialylated N-glycopeptide Sr-GP2, where up to four acetyl groups are detected.

Glycosylation of major N-glycoproteins in salmon skin
N-glycosylated proteins in the salmon skin extract were isolated by concanavalin A (ConA) affinity chromatography. The glycoprotein fraction was further separated by RP chromatography, yielding a major peak carrying two components of apparent MW 80 and 55 kDa in SDS–PAGE (not shown). These were subjected to in-gel digestion and identification by mass mapping. The 80-kDa protein was identified as transferrin. The 55-kDa protein (GP55) could not be identified by mass mapping, nor did N-terminal sequencing of five tryptic peptides reveal clear homology to any known protein.

An aliquot of the RP-fraction carrying transferrin and GP55 was subjected to N-glycan liberation, and the isolated N-glycans were analyzed by MALDI-TOF MS. The spectrum was very similar to that obtained from salmon kininogen and salarin N-glycans (not shown), indicating extensive sialic acid O-acetylation also in the N-glycans of these major glycoproteins of salmon skin.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In the present study we describe glycan structures present in two cysteine proteinase inhibitors of Atlantic salmon skin. Both glycoproteins were found to carry biantennary {alpha}2,3-sialylated N-glycans, as well as O-glycosidic sialylated core type 1 tri- and tetrasaccharides. The most interesting feature observed was that the N-glycans from these proteins, as well as those from the major N-glycoproteins present in salmon skin, exhibited very highly O-acetylated sialic acids. In fact, O-acetylated sialic acids are practically the sole terminal units in the N-glycans, as insignificant amount of non-O-acetylated N-acetylneuraminic acid is observed even after prolonged exposure to pH 8 during the protein purification (Ylönen et al., 1999Go). To our knowledge, these structures represent the most extensively O-acetylated N-glycans reported on any species.

Few detailed studies on salmonid N-glycosylation have been presented. Rainbow trout ovarian fluid contains a 54-kDa protein that carries very large tetraantennary N-glycans with I antigenic poly-N-acetyllactosaminoglycans, Lewis X, and {alpha}2,8-disialyl groups (Funakoshi et al., 1997Go). Atlantic salmon antithrombin has been found by lectin blotting studies to carry complex-type, {alpha}2,3-sialylated N-glycans (Andersen et al., 2000Go). No sialic acid O-acetylation was reported in these studies. However, the presence of O-acetylated sialic acids in salmon antithrombin can not be excluded, as {alpha}2,3-sialylation was identified by Maackia amurensis lectin, which binds to {alpha}2,3-linked sialic acids irrespective of their side chain O-acetylation status (Varki, 1997Go).

Interestingly, we have recently found high levels of sialic acid O-acetylation in the skin glycoproteins of two other northern Atlantic fish species, spotted wolffish (Anarhichas minor) and cod (Gadus morhua L.) (Ylönen et al., unpublished results). Atlantic salmon as well as other salmonids are considered primitive members of the teleost fishes. The conservation of extensive O-acetylation patterns in more evolved species, like spotted wolffish, may indicate an important function for this sialic acid modification in these species. General protection against microorganisms is one possibility, as O-acetylation of sialic acids is known to confer partial or complete resistance against sialidases and also to mask attachment sites on cells used by several bacteria and viruses (Varki, 1992Go).

The liver is the major site of kininogen expression in many species (Müller-Esterl et al., 1986Go). Moreover, salarin seems to be expressed mainly in the liver (Ylönen et al., unpublished results). Transferrin, which is mainly expressed in liver as well (Kvingedal et al., 1993Go), was found to carry similar levels of O-acetylation, and thus it is probable that the very effective sialic acid O-acetylation machinery resides in the salmon liver. This agrees well with earlier studies on sialic acid O-acetylation of N-glycans in rats (Butor et al., 1993Go). The O-acetyltransferases of salmon liver may however be distinct from those in rat liver, where mainly {alpha}2,6-sialylated N-glycans are O-acetylated (Butor et al., 1993Go).

We noted that two O-acetyl groups at most were present in the disialylated O-glycopeptide (Sr-GP1) isolated from salarin. Substantial loss of O-acetyl groups is unavoidable during the trypsin digestion, but a series of acetylated species carrying up to four O-acetyl groups were still detected in the disialylated N-glycopeptides. The overall acetylation efficiency of O-glycans may thus be lower. Alternatively, it is possible that either one of the sialic acids in the disialylated O-glycan is not a target for the O-acetyltransferase(s). This question however could not be addressed in more detail by the methodology used in this study: There is no practical method to liberate O-glycans from proteins without destroying their O-acetyl substituents. Nuclear magnetic resonance (NMR) is capable of distinguishing substituted sialic acids in branched glycans (Damm et al., 1989Go): Larger amounts of glycopeptides should therefore be prepared for NMR analysis to resolve whether there is preference for the sialic acid O-acetylation in O-glycans.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Salmon kininogen and salarin were purified from the skin of Atlantic salmon as described elsewhere (Ylönen et al., 1999Go).

Preparation of N-glycans
N-glycans from salmon kininogen and salarin samples were released by enzymatic hydrolysis with PNGase F, and purified as described (Saarinen et al., 1999Go). The oligosaccharides were recovered as a pool, and no separation of the individual components was attempted prior to MS analyses. The amount of N-glycans was estimated by their UV absorbance in gel filtration chromatography against external N-acetylglucosamine and N-acetylneuraminic acid standards.

The major glycoproteins of the salmon skin extract were isolated by affinity chromatography on a 5 x 45 mm column of ConA sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Samples of the delipidated skin extract were first run on a Bio-Gel P-6 DG column (10 x 100 mm) in 20 mM Tris–HCl, 250 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 (ConA buffer) to remove saccharose present in the extract, and then aliquots of 1–3 ml were applied to the ConA column. Nonbinding proteins were removed by washing with five column volumes of the ConA buffer, and the N-glycosylated proteins were then eluted with 5 ml of 0.5 M {alpha}-methylmannoside in ConA buffer. Pooled glycoprotein fractions were concentrated with pressure microfiltration (Omegacell 10K, Pall Filtron, Northborough, MA) and separated on a C1 RP chromatography column, yielding a major peak carrying two glycoproteins of 80 kDa and 55 kDa, as evident on SDS–PAGE (not shown). An aliquot of the RP-chromatography fraction (approximately 40 µg total protein) was vacuum-dried and then subjected to PNGase F treatment without attempts to separate the two components.

Preparation of O-glycans
To liberate O-glycosidically linked glycans, vacuum-dried protein samples obtained from RP-chromatography were dissolved in 100 µl of 1 M NaBH4 in 0.1 M NaOH, and incubated for 48 h at 37°C. The liberated oligosaccharide alditols were recovered as described (Nyman et al., 1998Go).

Permethylation
Vacuum-dried samples were permethylated according to the method of Ciucanu and Kerek (1984)Go. Prior to ESI-MS analyses, the permethylated glycans were purified by adsorption to POROS R3 material (Perseptive Biosystems, Framingham, MA) as follows (Saarinen et al., 1999Go): about 1 µl of POROS R3 material was packed into an Eppendorf gel-loader tip and washed with water, then the permethylated glycans were applied to the tip in 20% aqueous methanol. The column was washed with 20 µl of 10% aqueous methanol and eluted with 5 µl of 50% aqueous methanol.

Removal of O-acetyl groups
To remove O-acetyl groups from the isolated N-glycans, dry glycan samples were dissolved in 0.1 M NaOH and incubated at 8°C for 80 min. Reaction was terminated by adding one-half volume of 1 M acetic acid. An aliquot of the reaction mixture was drop-dialyzed for 30 min against water on VSWP 02500 membranes (Millipore, Bedford, MA) prior to MALDI-TOF MS analysis.

Periodate oxidation
Saponified N-glycans were incubated in 65 µl of 5 mM NaIO4 on ice in the dark for 15 min, and the excess of periodate was destroyed by addition of 10 µl of 0.3 M ethylene glycol (Van Lenten and Ashwell, 1971Go). After 30 min at room temperature, 50 µl of 1 M NaBH4 in 0.2 M Na2CO3 was added and the mixture was incubated at room temperature for 4 h. The reaction was terminated by adding 2 M acetic acid, and prior to MALDI-TOF MS analysis the N-glycans were desalted by gel filtration.

Gas-phase ß-elimination
Glycopeptide sample was dried in a glass tube, and the tube was placed in a larger glass tube containing 300 µl of 40% aqueous methylamine (Mirgorodskaya and Roepstorff, 1998Go). The tube was evacuated until cool to the touch, and the sample was allowed to react with methylamine gas for 2.5 h at 55°C. The deglycosylated peptide was dissolved in 50% aqueous acetonitrile, and an aliquot was analyzed by MALDI-TOF MS.

Glycosidase digestions
Liberation of N-glycans from N-glycopeptides was performed by enzymatic hydrolysis with PNGase F: Glycopeptide samples were dissolved in 30 µl of 20 mM sodium phosphate, pH 7.2, containing 0.2% ß-octylglucoside, and 1.5 U of PNGase F was added. After 16 h incubation at 37°C, a sample of the mixture was purified on a 1 µl column of POROS R3 as above, and the deglycosylated peptide was analyzed by MALDI-TOF MS.

Digestions with NDV neuraminidase and S. pneumoniae ß1,4-galactosidase (Oxford Glycosciences, Abingdon, UK) were carried out in 10 µl of 50 mM sodium acetate, pH 5.5, at 37°C; aliquots of 1 µl were removed after 16 h of digestion, drop-dialyzed against water, and analyzed by MALDI-TOF MS. Pure oligosaccharide standards were used as controls for the glycosidase reactions: Neu5Ac{alpha}2,3Galß1,4(Fuc{alpha}1,3)GlcNAcßOMe for NDV neuraminidase, and Galß1,4GlcNAcß1,6[Gal-ß1,3GlcNAcß1,3]Galß1,4Glc for ß1,4-galactosidase.

Trypsin digestion
Protein samples (20–40 µg) were alkylated with 4-vinylpyridine and desalted by RP-HPLC as described elsewhere (Ylönen et al., 1999Go). The vacuum-dried samples were dissolved in 30 µl of 0.1 M ammonium bicarbonate, and 0.5 µg of modified trypsin (Promega, Madison, WI) was added. Digestion was performed overnight at 37°C. The tryptic peptides were separated on a narrow-bore RP chromatography as described (Ylönen et al., 1999Go), and samples from the fractions were analyzed by MALDI-TOF MS.

MS
MALDI-TOF MS was performed on a BiflexTM time-of-flight instrument (Bruker Daltonik, Bremen, Germany) equipped with a nitrogen laser operating at 337 nm.

Underivatized N-glycans were analyzed in either positive ion delayed extraction reflector mode or negative ion delayed extraction linear mode, using 2,4,6-trihydroxyacetophenone (Fluka Chemie AG, Buchs, Switzerland) (3 mg/ml in acetonitrile/20 mM aqueous diammonium citrate, 1:1, by volume) as the matrix as described (Saarinen et al., 1999Go). Permethylated samples were analyzed in the positive ion delayed extraction reflector mode using 10 mg/ml DHB (Aldrich, Steinheim, Germany) in H2O as the matrix.

Peptide samples were analyzed using {alpha}-cyano-4-hydroxycinnamic acid matrix, as described elsewhere (Saarinen et al., 1999Go). Mass values for glycopeptides are average; for deglycosylated peptides monoisotopic values were obtained.

ESI mass spectra were collected using a Q-TOF hybrid quadrupole-orthogonal acceleration time-of-flight mass spectrometer (Micromass Ltd., Manchester, UK). Permethylated glycans were dissolved in 50% aqueous methanol containing either 0.5 mM NaOH (O-glycans) or 5 mM ammonium acetate (N-glycans) and injected into the mass spectrometer with a nanoelectrospray ion source at a flow rate of about 30 nl/min. For MS/MS studies the parent ions were selectively transmitted and directed into the collision cell containing argon collision gas. Collision energies were about 60 V for the sodiated singly charged permethylated O-glycans, and about 35 V for the ammoniated doubly charged permethylated N-glycans.

Characterization of O-acetylated sialic acids by LC-MS/MS
Sialic acids were liberated from salmon kininogen and salarin by chemical hydrolysis in 2 M propionic acid (Mawhinney and Chance, 1994Go). The released sialic acids were purified from proteins by running the hydrolysate through a POROS R1 microcolumn as above, and the eluate (25 µl) was mixed with 25 µl of DMB reagent to produce the corresponding sialic acid quinoxalinone derivatives (Hara et al., 1989Go; Klein et al., 1997Go). DMB-derivatized sialic acids were separated by microbore reversed-phase HPLC on an 0.075 x 150 mm PepMap column (NAN75-15-03-C18-PM; LC Packings, Amsterdam) by elution with a linear gradient of acetonitrile (4–40% in 30 min) in 0.1% formic acid. Chromatography was performed at a flow rate of 0.25 µl/min and the eluent was directly injected to Q-TOF mass spectrometer via a distally coated silica capillary needle (20 µm inner diameter, 10 µm tip opening; New Objective Inc., Cambridge, MA). The mass spectrometer was set to scan the HPLC eluent using the following settings: The MS was set to operate in constant CID mode for the entire HPLC run, with the MS set to acquire CID spectra on ions 426.2, 468.2, and 510.2 Da, corresponding to [M+H]+ of non-O-acetylated, mono-O-acetylated, and di-O-acetylated DMB-N-acetylneuraminic acid, respectively. Each spectra were acquired for 1.5 s/parent ion, ramping between the selected ions for the entire HPLC run. The collision energy for all ions was 18 V.

Protein identification by mass mapping
Protein samples were run on SDS–PAGE; bands of interest were cut out of the gel and subsequently hydrolyzed in-gel with trypsin. An aliquot of the peptide mixture obtained was desalted on a POROS R3 microcolumn and analyzed by MALDI-TOF MS as described (Nyman et al., 2000Go).

Peptide sequencing by Edman degradation
Peptides were sequenced using a Procise 494A sequencer (Perkin-Elmer Applied Biosystems Division, Foster City, CA).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The authors wish to thank Ms. Anu Jaakola, Mrs. Gunilla Rönnholm, and Mr. Keijo Virta for excellent technical assistance. This study was partly supported by grants from the Academy of Finland (no. 41413 and no. 46692 to J.H.). This study was also partly supported by the Commission of the European Communities, Agriculture and Fisheries specific RTD program CT97-3508, "Fish cysteine proteinase inhibitors and infectious diseases"; it does not necessarily reflect the commision’s views and in no way anticipates its future policy in this area.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ac, acetyl; ConA, concanavalin A; DMB, 1,2-diamino-4,5-methylenedioxybenzene; ESI, electrospray ionization; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MW, molecular weight; NDV, Newcastle disease virus; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide:N-glycosidase F; RP, reversed-phase; SK-GP, glycopeptide from salmon kininogen; Sr-GP, glycopeptide from salarin.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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