Involvement of glycan chains in the antigenicity of Rapana thomasiana hemocyanin

Biochemical and Biophysical Research Communications 361 (2007) 705–711 www.elsevier.com/locate/ybbrc

Involvement of glycan chains in the antigenicity of Rapana thomasiana hemocyanin Nurul Islam Siddiqui a, Krassimira Idakieva b, Bert Demarsin c, Lyubka Doumanova d, Frans Compernolle c, Constant Gielens a,* a

c

Division of Biochemistry, Molecular and Structural Biology, Chemistry Department, Katholieke Universiteit Leuven, Celestijnenlaan 200 G, 3001 Leuven-Heverlee, Belgium b Institute of Organic Chemistry, Bulgarian Academy of Sciences, Akad. G. Bonchev-Str. bl. 9, Sofia 1113, Bulgaria Division of Molecular Design and Synthesis, Chemistry Department, Katholieke Universiteit Leuven, Celestijnenlaan 200 F, 3001 Leuven-Heverlee, Belgium d Institute of Microbiology, Bulgarian Academy of Sciences, Akad. G. Bonchev-Str. 26, Sofia 1113, Bulgaria Received 17 July 2007 Available online 26 July 2007

Abstract Functional unit (FU) RtH2-e from Rapana thomasiana hemocyanin (Hc) was degraded into small fragments with chymotrypsin. The glycopeptides were separated from the non-glycosylated peptides by chromatography on Concanavalin-A–Sepharose and characterized by mass spectrometry. The glycan part of the glycopeptides (all with common peptide stretch of 14 amino acids) consists of the classical trimannosyl-N,N-diacetylchitobiose core for N-glycosylation, predominantly extended with a unique tetrasaccharide that is branched on fucose. In inhibition ELISA experiments, the glycopeptides interfered in the complex formation between FU RtH2-e and rabbit antibodies against Rapana Hc (about 30% of inhibition). The inhibition also was retained after treatment of the glycopeptides with pronase in order to completely destroy the peptide part. The inhibitory effect of the non-glycosylated peptides, on the other hand, was very low. This study thus demonstrates that the glycans attached to FU RtH2-e contribute to the antigenicity of Rapana Hc.  2007 Elsevier Inc. All rights reserved. Keywords: Antigenicity; Electrospray ionization-mass spectrometry; ELISA; Gastropod; Glycan; Rapana thomasiana; Hemocyanin; Mollusc

The hemocyanins (Hcs) are extracellular type-3 copper proteins occurring in high concentration in the hemolymph of molluscs and arthropods. Molluscan Hcs exist as cylindrical structures that are built up of 10 (cephalopods) or 20 (gastropods) 350–450 kDa subunits. These subunits are composed of seven or eight globular functional units (FUs) denoted by the letters a to g (or h) read from the N-terminus on. Each FU has an average molecular mass of about 50 kDa and carries a pair of copper atoms enabling the reversible binding of molecular oxygen [1,2]. In addition to their function as oxygen transporting proteins, Hcs play a role in the innate immune system of the animals as they possess some phenoloxidase activity, which can be enhanced e.g. by limited proteolysis [3,4]. *

Corresponding author. Fax: +32 16 327978. E-mail address: [email protected] (C. Gielens).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.098

Hcs are able to stimulate the immune system in many organisms including human. In particular, this was studied for the Hc of the marine gastropod Megathura crenulata, commonly known as keyhole limpet hemocyanin (KLH), which has frequently been used as an immunotherapeutic agent in the treatment of certain kinds of cancer (mainly bladder carcinoma) and as a carrier for vaccines [5]. Hc from another marine gastropod, Haliotis tuberculata is considered to be a possible substitute for KLH as immunostimulant [6]. Recently, also the Hc from a Chilean gastropod, Concholepas concholepas [7], was shown to have significant antitumor activity against mouse bladder carcinoma cells [8]. Studies, basically made on KLH, showed that the glycan structures present on the protein play a significant role in the antigenicity [5]. These glycans are more abundant on molluscan Hcs (2–6% carbohydrate, w/w) than

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on arthropodan Hcs [9]. Within the molluscan phylum, gastropodan Hcs are richer in sugar than cephalopodan ones [9]. The successful use of KLH as antitumor agent and vaccine carrier and the growing evidence for the antigenic potential of its sugar motifs have made it interesting to explore the carbohydrate based antigenicity of other related Hcs, especially from gastropods. In this context, our present study deals with the antigenicity of the Hc of Rapana thomasiana (Rt), a marine gastropod living along the west coast of the Black Sea. This Hc exists in the hemolymph as two distinct isoforms, RtH1 and RtH2, both characterized by the typical quaternary structure of gastropodan Hcs [10] and a carbohydrate content of 2.6% (w/w) [11]. For FU RtH2-e (i.e. FU e from the second isoform), the primary structure [12] and crystal structure (PDB code 1LNL) [13] are known. Recently, our group characterized the N-linked glycans of this FU [14]. A novel motif was discovered, consisting of a central fucose moiety that is substituted with 3-O-methylgalactose and N-acetylgalactosamine and linked to Nacetylglucosamine at the reducing end and that is further connected to one of the mannose arms of the core structure. The purpose of this work was to study the involvement of this peculiar glycan structure in the antigenicity of Rapana Hc. Materials and methods Chymotrypsinolysis of functional unit RtH2-e and separation of fractions. FU RtH2-e was isolated from R. thomasiana Hc [15] and next denatured, reduced and S-pyridylethylated as described [14]. After dialysis against 0.1 M NH4HCO3, pH 8.2, the solution (7 mg in 2 mL) was treated with a-chymotrypsin (Sigma, St. Louis, MI, USA) at an enzyme/ substrate ratio of 1/50 (w/w) for 18 h at room temperature. The digest was applied on a column (9.6 · 0.9 cm) of Concanavalin-A–Sepharose 4B (Sigma), equilibrated with binding buffer (20 mM Tris/HCl, pH 7.4, containing 0.5 M NaCl, 1 mM MnCl2 1 mM CaCl2, and 1 mM MgCl2). The column was eluted with binding buffer for recovery of the non-glycosylated peptides, and subsequently with the same buffer containing 0.5 M methyl-a-D-glucopyranoside for desorption of the glycopeptides. Determination of (glyco)peptide concentration. The concentration of the peptide and glycopeptide fractions (expressed in lg peptide/lL) was estimated by amino acid analysis on a 4151 Alpha Plus amino acid analyzer (LKB, Uppsala, Sweden), equipped with an Alpha Plus PEEK column. For the glycopeptide fraction the mass of the bound carbohydrates is thus not taken into account. The hydrolyses were performed in the presence of norleucine (5 nmol) as internal standard with azeotropic HCl, 0.1% phenol, in evacuated sealed tubes at 110 C for 24 h. Liquid chromatography/electrospray ionization-mass spectrometry. The LC system was equipped with an Agilent 1100 binary pump, degasser, and autosampler. A C-18 Alltech Prevail 5 lm LC column (150 · 2.1 mm) was used. Ten microliters of sample (glycopeptide fraction from ConcanavalinA chromatography; peptide concentration 0.02 lg/lL) was injected in the LC apparatus and a linear gradient of acetonitrile (from 10% to 80% in 30 min) in 0.1% aqueous HCOOH was applied. The flow rate was maintained at 0.2 mL/min. The system was directly coupled with a LCQ Advantage ion trap mass spectrometer of Thermo Finnigan (San Jose, CA, USA) equipped with an ESI (electrospray ionization) source operating in the positive-ion mode to generate molecular ions (M+xH)x+ of the glycopeptides. Besides full MS scans (upper mass limit m/z 2000), also MS/MS spectra were recorded. Spray voltage was maintained at

approximately 4.5 kV, and N2 was used as a nebulizing gas. The collision energy was set as 25%. Preparation of antiserum. The antiserum against total Rapana Hc was prepared according to the protocol described in [16]. For the ELISA experiments the antiserum (anti-Rapana Hc) was diluted in phosphatebuffered saline (PBS). Competitive enzyme linked immunosorbent assay. For each competitive ELISA experiment two microtiter (96 wells) plates were used. A first plate was coated with the antigen by application of a solution (100 lL per well) of FU RtH2-e at a concentration of 50 nM in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3), pH 9.7, at 4 C overnight. The plate was rinsed three times with wash buffer (0.05% Tween 20 in PBS), treated for 1 h at room temperature with a 1% casein solution in PBS (200 lL per well) to block the unoccupied places and rinsed three times with wash buffer. A second plate was coated with 1% casein solution (200 lL per well) for 1 h at room temperature. This plate was used for pre-incubation of the antibodies with the potential competitors (peptide and glycopeptide fractions) in the reaction with FU RtH2-e. In the classical competitive ELISA experiments, 158 lL of antiserum, 500· diluted in PBS, was added in the first well of the row and a semilogarithmic dilution series was made by transferring 50 lL to the successive wells filled with 108 lL PBS. Then to each well of a dilution series 0.2 lg peptide, i.e. 10 lL of (glyco)peptide fraction brought to a concentration of 0.02 lg peptide/lL with PBS, was added. In a second approach [17], increasing amounts (0.02–0.4 lg peptide; in a final volume of 20 lL) were added to 108 lL of 5000· diluted antiserum (corresponding to the third point in the dilution series in the first approach). In parallel series FU RtH2-e was added instead of the (glyco)peptide fraction to obtain conditions of 100% inhibition. After 3 h at room temperature the mixtures were transferred to the wells of the antigen-coated plate (first plate). This plate was then incubated for 1 h at room temperature (for reaction of the free antibodies with the coated RtH2-e) and then rinsed with wash buffer. Next, 100 lL of secondary antibodies, namely goat anti-rabbit antibodies conjugated with alkaline phosphatase (GaR-AP) (ICN, Aurora, OH, USA), diluted to 1:10,000, v/v, in PBS with 0.1% casein, was added to each well of the plate. After 1 h at room temperature the plate was washed and 100 lL of para-nitrophenylphosphate dissolved at 0.5 mg/mL in 0.1 M glycine–NaOH buffer, pH 10.4, 1 mM ZnCl2, 1 mM MgCl2, was added per well as substrate. Finally, the plate was measured at 405 nm by a multichannel-spectrophotometer (ELX800, Bio-Tek Instruments, Winooski, VT, USA). The absorbance values were plotted against log10(dilution). Pronase treatment. The (glyco)peptide fractions (pH 7.4) were treated with pronase (Calbiochem, La Jolla, CA, USA) in an enzyme to substrate ratio of 1/1 (w/w) for 48 h at 37 C.

Results Separation and MS analysis of chymotryptic glycopeptides In our earlier work, making use of trypsin, the glycans attached to FU RtH2-e were determined [14]. Four main glycopeptides, Gp Ia, Gp Ib, Gp IIa, and Gp IIb, all with the same peptide part (52 amino acids long; peptide stretch 92–143 in the sequence of RtH2-e) were found. The glycan structures, deduced by a thorough MS and MS/MS analysis, are schematised in Fig. 1A. As the peptide part of these glycopeptides could interfere in the study of the antigenic properties of the glycans, we now used chymotrypsin instead of trypsin to degrade FU RtH2-e so as to obtain glycopeptides with shorter peptide stretch. The glycopeptides (ConA+ fraction) were separated from the non-glycosylated peptides (ConA fraction) as described in Materials and methods. On LC/ESI-MS of the ConA+ fraction four main elution peaks were observed

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707

Fig. 1. Glycopeptides of functional unit RtH2-e from Rapana Hc. (A) N-Glycan structures as deduced by MS/MS analysis of tryptic glycopeptides [14]. Glycopeptides used in the present study were obtained with chymotrypsin and were termed Gp Ia 0 , Gp Ib 0 , Gp IIa 0 , and Gp IIb 0 . The nomenclature for the glycan fragmentations on MS/MS is according to [18]. P, peptide part. Symbols for monosaccharides: v, fucose; s, mannose; d, 3-O-methylgalactose; h, N-acetylgalactosamine; j, N-acetylglucosamine. (B). Peptide stretch in the vicinity of the glycan attachment site (Asn-127; in box) with main (full arrows) and minor (broken arrows) chymotrypsin cleavage sites.

(Fig. 2A). MS analysis of fraction I (retention time on LC 12.1–12.3 min) (Fig. 2B) revealed two main components: a major one (Gp Ia 0 ) with m/z 1678.0 and a minor one (Gp Ib 0 ) with m/z 1779.1. Likewise, two main peaks (Gp IIa 0 and Gp IIb 0 ) appeared in the MS scan of fraction II (retention time 12.5–12.6 min) (Fig. 2C). The chymotryptic glycopeptides were designated with a prime in order to distinguish them from the tryptic ones. On subtracting the glycan parts, a same molecular mass (1732 Da) was obtained for all chymotryptic glycopeptides (Table 1), showing that they all share the same peptide part. The glycan mass values of the tryptic counterparts were used for this calculation. The peptide stretch was localized in the sequence of RtH2-e (Fig. 1B), taking into account the potential chymotrypsin cleavage

sites in the FU [12]. The theoretical mass of the peptide (stretch 115–128 in the sequence of RtH2-e) amounts to 1732.8 Da, which is in good agreement with the experimentally deduced value (Table 1). MS2 analysis of the molecular ions corresponding to Gp 0 Ia , Gp Ib 0 , Gp IIa 0 , and Gp IIb 0 , supplemented with MS3 analysis of selected fragment ions, confirmed the glycan structures deduced in our earlier work from the results with the tryptic glycopeptides [14] (see also Fig. 1A). As an example the MS2 spectrum of the predominant glycopeptide Gp Ia 0 is shown in Fig. 3, the interpretation of the data is given in Table 2. The MS spectrum of fraction III (Fig. 2A) revealed molecular ions at m/z 737.0 ([M+2H]2+) and 1471.7 ([M+H]+), indicating a molecular mass of 1471.3 Da

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Relative abundance

A

60

40

20

0 10

B

11

12

100

13 Time (min)

14

15

16

1678.0

Relative abundance

80 60 40 1779.1

20 0 1500

1600

1700

1800

1900

2000

m/z

C

100

1313.8

Relative abundance

80 1414.9

60 40 20 0 1200

1300

1400

1500

1600

1700

m/z Fig. 2. LC/ESI-MS of the chymotryptic glycopeptides of RtH2-e. (A) Base-peak chromatogram of the RtH2-e glycopeptide fraction (ConA+) showing four prominent peaks. The first two peaks, designated by I and II, are analogous to the HPLC peaks (I and II) of the tryptic glycopeptides of RtH2-e (earlier work, [14]). Fractions III and IV appeared to correspond to the peptide part of the glycopeptides after C-terminal loss of two and four amino acids, respectively (see Fig. 1B). (B,C) Mass spectra of fractions I and II (see also Table 1).

(average of 1472.0 and 1470.7 Da obtained for the doubly and singly charged ions, respectively). The spectrum of fraction IV (Fig. 2A) showed a signal at m/z 1263.6 ([M+H]+), indicative of a molecular mass of 1262.6 Da. From these data and amino acid sequence information obtained from further MS/MS analysis of the singly charged molecular ions it could be concluded that frac-

tion III corresponds to peptide stretch 115–126 (theoretical mass 1471.5) and fraction IV to peptide stretch 115–124 (theoretical mass 1263.3) of RtH2-e. Fractions III and IV thus result from the C-terminal loss of respectively a dipeptide and a tetrapeptide, both containing the glycan part of the glycopeptides (see Fig. 1B).

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709

Table 1 Identification by LC/ESI-MS of glycopeptides obtained by chymotrypsinolysis of functional unit RtH2-e Retention time on LC (min)

Glycopeptide m/z

Molecular mass (Da) Molecular ion 2+

Designation

Total

Glycan parta

Peptide partb

12.1–12.3

1678.0 1779.1

[M+2H] [M+2H]2+

Gp Ia 0 Gp Ib 0

3354.0 3556.2

1621.6 1824.9

1732.4 1731.3

12.5–12.6

1313.8 1414.9

[M+2H]2+ [M+2H]2+

Gp IIa 0 Gp IIb 0

2625.6 2827.8

892.9 1096.1

1732.7 1731.7

a b

Values as observed for the corresponding tryptic glycopeptides; taken from [14]; for oligosaccharide structures, see Fig. 1. Obtained by subtraction of the glycan part from the total molecular mass value.

2+

Y3β 1232.6

100

% Intensity

80

2+

60 2+ 2+

Y4β

Y1

40

968.8

Y5βY3α 1333.9

2+

Y6γ

1313.9

2+

Y3αY3β 2+

Y5β

1151.1

Y2

20

1415.4

1071.0

0 900

1000

1100

1576.6

2+ 2+

Y6βY6γ 1489.0

1200

1300

1400

2+

Y6β 1589.7

1500

1600

1700

m/z Fig. 3. MS2 analysis of chymotryptic glycopeptide Gp Ia 0 (molecular ion [M+2H]2+ with m/z 1678.0), showing the glycan fragmentations. Assignments are given in Table 2.

Table 2 MS2 analysis of the molecular ion [M+2H]2+ generated from glycopeptide Gp Ia 0 (m/z = 1678.0) m/z

1589.7 1576.6 1489.0 1415.4 1333.9 1313.9 1232.6 1151.1 1071.0 968.8

Assignment of ions [Y6bP]2+ [Y6cP]2+ [Y6bY6cP]2+ [Y5bP]2+ [Y5bY3aP]2+ [Y4bP]2+ [Y3bP]2+ [Y3aY3bP]2+ [Y2P]2+ [Y1P]2+

Molecular mass (Da)

Molecular composition

Remaining

Lost

3177.4 3151.2 2976.0 2828.8 2665.8 2625.8 2463.2 2300.2 2135.4 1935.6

176.6 202.8 378.0 525.2 688.2 728.2 890.8 1053.8 1218.6 1418.4

P-HexNAc4-Man3-Fuc P-HexNAc3-Man3-Fuc-3MeGal P-HexNAc3-Man3-Fuc P-HexNAc3-Man3 P-HexNAc3-Man2 P-HexNAc2-Man3 P-HexNAc2-Man2 P-HexNAc2-Man P-HexNAc2 P-HexNAc

The nomenclature for the glycan fragmentations is according to that introduced by Domon and Costello [18]; see also Fig. 1.

Contribution of glycans to the antigenicity In order to study the involvement of the bound glycans in the antigenicity of RtH2-e, and thus of Rapana Hc, inhibition ELISA experiments were performed with a dilution

series of the antiserum (against total Rapana Hc) preexposed to either the chymotrypsin generated glycopeptide fraction (ConA+) or the non-glycopeptide fraction (ConA). The results show a definitely higher level of inhibition by the ConA+ fraction than by the ConA fraction

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(Fig. 4A). In order to exclude the contribution of the peptide part (which is still 14 amino acids long for the glycopeptides), both the ConA+ and ConA fractions were treated with pronase and were then allowed to react with antiserum. In this experiment the inhibitory effect of the ConA+ fraction was retained, clearly indicating a contribution of the glycan structures of RtH2-e in the observed immunoreactivity. The inhibitory effect of the RtH2-e glycans was also demonstrated by inhibition ELISA experiments performed with increasing amounts of peptide/glycopeptide. In this case the antiserum was used at a single concentration in the successive wells of the reaction series. The antiserum concentration was chosen at a particular dilution shown to give a medium level of absorbance in the regular inhibition ELISA experiments (Fig. 4A). Again, a clear inhibitory effect of the ConA+ fraction in the complex formation between RtH2-e and the antibodies was observed; in contrast, the ConA fraction gave nearly no inhibition as indicated by the flat top line (Fig. 4B). On considering the inhibition by the complete FU

Fig. 4. Competitive ELISA using the glycopeptide (ConA+) and nonglycopeptide (ConA) fractions as potential inhibitors in the reaction between functional unit RtH2-e and antibodies against Rapana Hc. (A) Classical experiment employing a dilution series of the antiserum and 0.2 lg peptide or protein (RtH2-e). Blank curve represents ELISA without any inhibitor added. (B) Experiment with increasing amounts of peptide (or RtH2-e) at a same antiserum dilution. Absorbance values are means of four independent determinations; vertical bars represent the standard deviation. In all cases microtiter plates were coated with RtH2-e (50 nM).

RtH2-e as 100%, about 30% inhibition was calculated for the ConA+ fraction. The graph also shows the minimum amount of peptide (as inhibitor) needed for the optimum complexation with the antiserum. This is about 0.1 lg/well in this experiment. In the regular inhibition experiments (Fig. 4A), we applied 0.2 lg of peptide per well which thus is sufficient for the complexation with the antibodies. Discussion So far the antigenicity and therapeutic potency of Hcs are best documented for KLH (e.g., [5,6,19]). These properties of KLH are in part ascribed to the bound sugar components, which comprise next to O-glycans that are only present on FU KLH2-c [20,21], also a variety of N-glycans [22–25]. Recently, the Hc from the Chilean marine gastropod C. concholepas was reported to be as effective as KLH for preventing tumor growth in a murine bladder cancer model [8]. Since R. thomasiana belongs to the same family as C. concholepas (order Neogastropoda, family Rapaninae) its Hc is likely to show similar immunogenic properties. The LC/MS analysis of the chymotryptic glycopeptide fraction of FU RtH2-e fully confirmed the previously deduced N-glycan structure with the peculiar tetrasaccharide unit (see Fig. 1A). The mass spectral results also show that the glycopeptides were partially further cleaved near the C-terminal end (Fig. 1B). Presumably, the split peptides stick together during the ConA–Sepharose chromatography and are only separated at the time of the mass spectrometric analysis due to the acid conditions used for the LC. The released glycan-containing di- and tetrapeptides probably elute at the void volume of the LC column due to their expected hydrophilicity. Although the polypeptide chains of the gastropodan Hcs likely play a major role in the immunomodulatory function [8], only very little inhibition was noted in our experiments with the mixture of the non-glycosylated chymotryptic peptides. This could be due to the fact that the peptide stretches are too short and/or that the peptide epitopes were broken by the proteolysis. The observed inhibition of about 30% by the glycopeptide fraction would imply that nearly one-third of the antibodies with which the complete FU RtH2-e can react are specifically directed against the unique tetrasaccharide structure. In the current work, we thus provided further evidence for the participation of glycans in the antigenicity of gastropodan Hcs. It is most probable that in Rapana Hc, in addition to the ones studied here, other glycans act as carbohydrate epitopes. Indeed, e.g., 3-O-methylgalactose and sulphated mannose have been reported to form part of N-glycan structures on FU RtH1-a [26]. It is worth considering Rapana Hc as an alternative for KLH and thus to conduct experiments towards immunotherapeutic applications.

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Acknowledgments We thank the Fund for Scientific Research, Flanders (Belgium) and the Bulgarian Academy of Sciences for financial support of the joint research project ‘‘Conformational stability, antigenicity and enzymatic properties of gastropodan hemocyanins’’. This work was also supported by research Grant TK-X-1611 from the NSF of the Ministry of Education and Science, Bulgaria. We are grateful to the Katholieke Universiteit Leuven (Belgium) for an IRO fellowship (N.I.S.). F.C. thanks the Fund for Scientific Research, Flanders (Belgium) for financial assistance in acquiring the mass spectrometric equipment.

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