Characterization of Haptoglobin in the Blood Plasma of Harbor Seals (Phoca vitulina) Henning Rosenfeld, Stephan Lassen, and Andreas Prange* GKSS Research Centre, Institute for Coastal Research, Department for Marine Bioanalytical Chemistry, Max-Planck-Strasse 1, D-21502 Geesthacht, Germany Received January 14, 2009
Haptoglobin (Hp) is one of the acute phase proteins (APP) in the blood of vertebrates that is involved in immune responses. Hp concentrations are found to vary under conditions of, for example, infection, trauma or cancer. These variations and the changes in its constitution are frequently used to assess the health status of mammals. In this work, Hp from the blood plasma of diseased and healthy harbor seals was isolated and structurally characterized. The process developed for the isolation of Hp is based on glycoprotein enrichment from crude plasma samples by means of ConA lectin affinity separation. Structural features of the protein backbone and the N-glycans were determined using MALDI-TOF/ TOF-MS. De novo sequencing of seal Hp revealed an R-chain composed of 84 amino acids and a β-chain comprising 245 amino acids. Comparison with Hp of the phylogenically related dog and human Hp 1-1 reveals the conserved and variable regions. All cysteine residues responsible for disulfide bonds and one glycosylation site have identical positions in the primary structures. Altogether, four possible glycosylation sites were identified. The glycoprofile is dominated by disialylated biantennary complextype glycans. Keywords: Acute phase protein • blood plasma protein • glycoprotein • haptoglobin • lectin separation • marine mammal health • harbor seals
1. Introduction
diagnostic tools for the monitoring of the health of the harbor seal population in the North Sea.
For decades, environmental assessment or the description of the status of a selected ecosystem has been based on the quantification of pollution levels of either organic or inorganic contaminants in different compartments of the environment or via measuring the body burdens of selected organisms.1–3 Recently, the assessment of biological responses using molecular biomarkers such as proteins, which are involved in biological key processes, has gained more interest.4,5 Methodological progress, especially within the field of proteomics, has opened up outstanding possibilities to propose new biomarkers on a molecular level, which might describe pollution-induced effects or the health status of an individual.6
The plasma proteome in mammals has a high potential for disease diagnosis7,8 and can also be utilized to identify suitable biomarkers that reflect the pathological status of an individual.9,10 During an acute phase response, which is an inflammatory reaction that occurs immediately after exposure to a triggering event, the concentration of several plasma proteins, called acute phase proteins (APPs) changes by up to several orders of magnitude. The haptoglobin (Hp) concentration in the blood plasma is elevated (positive APP) in the case of, for example, inflammation, lesions, infection or malignancy.11 A concise description of the interplay between organs and substances during an acute phase response in animals has been given by Petersen et al.12 Another function of Hp comprises the binding to hemoglobin (Hb).13,14 Under certain pathological conditions, the erythrocyte membrane may be destabilized, which results in hemoglobin release. The intrinsic peroxidase activity of Hb is capable of causing oxidative tissue damage.15 Hp builds a strong complex with Hb and inhibits this oxidative activity. Afterward, the complex is removed from circulation.14 Thus, Hp is involved in the repair and recovery of damaged tissue. Since Hp is synthesized mainly by hepatocytes, the concentration may further be diminished in the case of severe hepatocellular deficiencies.16
Marine mammal health is considered as an indicator for environmental changes in the marine ecosystem. Their position as top predators in the food web and their long life span is attended by a high bioaccumulation of pollutants. For the Wadden Sea ecosystem of Denmark, Germany, and The Netherlands, harbor seals are used within the Trilateral Monitoring and Assessment Program as a biological parameter with regard to the size of population and their reproduction rate. With respect to marine mammal health, however, diseases and their effects on the population require immunological and toxicological investigations, which can be used to develop * To whom correspondence should be addressed. Phone: +49(0)4152/ 87-1858. Fax: 04152-87-1875. E-mail:
[email protected]. 10.1021/pr900035s CCC: $40.75
2009 American Chemical Society
The biosynthesis of the glycans attached to the Hp protein backbone is subject to several influence capabilities, because glycosylation is a nontemplate driven, enzyme-guided process. Journal of Proteome Research 2009, 8, 2923–2932 2923 Published on Web 04/17/2009
research articles The final structure of the glycans attached to the protein depends on the expression (gene transcription) of the catalyzing enzymes (glycosyltransferases and glycosidases) and on their catalytic activity and proper location in the endoplasmatic reticulum as well as the Golgi apparatus. Consequently, the mammalian glycan expression can be influenced at either a post-transcriptional or a post-translational level.17 The importance of post-translational modifications and their modulating effects on protein function in health and disease has been described by Gabius, and Ohtsubo and Marth.17,18 However, the precise mechanism of impaired glycosylation caused by diverse diseases is not yet fully understood. Thibodeaux et al. reviewed investigations on the enzymes involved in the biosynthesis of glycans.19 It is generally accepted that the glycans have profound therapeutic and diagnostic potential20 and offer a new dimension for health assessment.21–23,16 Explaining how glycan diversity is regulated as a function of biosynthesis and understanding the characteristics and dynamics of posttranslational modifications are the most challenging tasks for the future.24 Hp glycan verifications in human medicine suggest that alterations in the glyco-structures are indeed indicative of several diseases.25–27 Haptoglobin can be used as a potential reporter molecule by exploiting its quantitative change11 as well as its varying glycosylation patterns.25 For some animals, the Hp structure is well-known. It often consists of R- and β-subunits that are connected via a disulfide bond (R-β). In humans, Hp has a tetra-chain alignment (βR-R-β).28 However, no data are available for the common seal. In this work, the Hp from harbor seals has been isolated and structurally characterized in order to form the basis for the development of a diagnostic tool for the monitoring of the health of the harbor seal population in the North Sea. Therefore, seals’ Hp had to be isolated from the plasma and de novo sequencing had to be performed with respect to its protein backbone as well as the analysis of the carbohydrate moieties.
2. Materials and Methods 2.1. Blood Samples from Seals. Seals from three different locations in the German and Danish Wadden Sea were surveyed for Hp investigations. First, blood samples from free ranging animals were collected, which were captured temporarily for monitoring purposes on the Lorenzenplate and the Island of Ro¨m, respectively. Afterward, they were released. Second, blood samples were taken from animals living in captivity in the Seal Station Friedrichskoog (Germany). Third, diseased or injured animals were investigated, which were euthanized by local seal rangers. Blood was sampled from the epidural vertebral vein. The whole blood samples were collected in EDTA monovettes from Sarstedt (Nu ¨ mbrecht, Germany) and transported to the laboratory at ambient temperature, where the samples were centrifuged at 3000g for 20 min. Aliquots of the blood plasma were stored at -80 °C until further analysis. 2.2. Haptoglobin Assay. Haptoglobin can be retraced during the isolation process with a colorimetric assay available from BioRepair (Sinsheim, Germany). The test exploits the intrinsic peroxidase activity of the Hb and its preservation in the Hb-Hp complex at low pH, which initiates a color reaction that can be detected at a wavelength λ ) 630 nm in a microplate reader. 2.3. Haptoglobin Isolation. Lectin adsorbents were prepared with Toyopearl AF-Tresyl 650 M (Tosoh Bioscience, Stuttgart, Germany) as solid support to which the lectin Concanavalin A (ConA) (obtained from Sigma Aldrich, Munich, 2924
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Rosenfeld et al. Germany) was immobilized. The reaction was completed after 4 h at pH 8 in 0.5 mol/L PBS. End-capping of excessive functional groups was performed with Tris buffer at the same pH. For detailed information about the features of the support and immobilization characteristics with lectin ligands, refer to Rosenfeld et al.29 For the adsorption of glycoproteins onto ConA-adsorbents, a 0.1 M acetate buffer (pH 6) containing Ca2+, Mn2+ and Mg2+ ions (2 mM) for the structural maintenance was used.30 Desorption occurred with 0.1 M R-methylmaltopyrannoside from Sigma Aldrich (Germany) dissolved in this acetate buffer. The enriched glycoproteins from the lectin separation were gel-filtrated using a Superdex 200 column (GE Healthcare, Freiburg, Germany). The column was equilibrated and proteins were dissolved in PBS pH 7.3, 0.15 M NaCl. The flow-rate was ¨ kta prime FPLC system with UV set to 0.3 mL/min on an A detection. The Hp containing fraction was applied to a weak anion exchanger DEAE FF (bed volume 5 mL) supplied by GE Healthcare (Freiburg, Germany). The loading of the samples occurred in Bis-Tris buffer pH 6.5 and a combination of a stepwise and linear gradient was applied to a final concentration of 1 M NaCl to elute bound proteins from the column. 2.4. Two-Dimensional Gel Electrophoresis (2-DE). The entire equipment and buffers used for the 2-DE was provided by BioRad (Munich, Germany). The protein separation based on its charge was carried out on IPG-strips with an immobilized pH gradient ranging from pH 3 to 10. The uptake of proteins, prior desalted by ultrafiltration, to the IPG strip was accomplished via active rehydration at low voltage (50 V) for 16 h in a Protean IEF cell. The following isoelectric focusing (IEF) was achieved with rising voltage gradients. Separation based on the protein size was accomplished in a Protean plus dodeca cell. Equilibration of the IPG strips was carried out in two buffers applied successively. The buffers react on the IPG strips for 15 min (both buffers contain 0.37 M Tris HCl, 6 M Urea, 2% (w/v) SDS, and 30% (v/v) glycerol; the first is spiked additionally with DTT and the second with iodacetamide (Sigma Aldrich, Germany)). The proteins were separated in a 12% polyacrylamide gel for ca. 24 h. Protein staining was conducted with Coomassie Brilliant Blue. 2.5. Preparation of the Protein Samples for Mass Spectrometry. 2-DE protein spots of the separated R and β chains were subjected to in-gel deglycosylation and enzymatic digestions with trypsin (Sigma Aldrich, Germany) and endoproteinase GluC (VWR, Darmstadt, Germany). Excised gel spots were incubated with 200 µL of destaining solution (40% acetonitrile in 200 mM ammonium bicarbonate) at 37 °C for 30 min. This step was repeated twice, and after discarding the supernatant, the gel spot was dried in a vacuum centrifuge for 30 min. For deglycosylation, 10 µL (5 units) of a peptide-N4-(N-acetylβ-D-glucosaminyl) asparagine amidase F (PNGaseF) solution supplied by Sigma Aldrich was added and the samples were incubated for 30 min at 37 °C. After covering the gel pieces with 20 µL of ultrapure water, the incubation was continued overnight for 17 h. The supernatant obtained from the PNGaseF digests was retained for the glycan analysis. The gel spots were washed with 200 µL of ultrapure water in an ultrasonic bath for 30 min three times and vacuum-dried. After adding 20 µL of trypsin (20 µg/mL 1 mM HCl) or alternatively endoproteinase GluC (20 µg/mL water) solution dissolved in an appropriate reaction buffer (50 µL of 40 mM ammonium bicarbonate containing 9% acetonitrile), the ingel enzymatic digestion proceeded overnight at 37 °C. The
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Characterization of Hp in the Blood Plasma of Harbor Seals resulting peptide solution was dried in a vacuum centrifuge. For MS analysis, the dried peptide mixture was redissolved in 10 µL acetonitrile/0.1% trifluoroacetic acid (TFA) (1:1, v/v). 2.6. MALDI TOF and TOF/TOF MS. R-Cyano-4-hydroxycinnamic acid (CHCA) was used as matrix. The solution was prepared by dissolving CHCA in an ethanol/acetone mixture (2:1, v/v) to a final concentration of 1 µg µL-1. Samples and matrix were premixed at a ratio of 1:1 and subsequently 1.5 µL of the mixture was applied to an 800 µm Anchor Chip (Bruker, Germany). Crystallization occurred at room temperature. Following an on-target washing step with 10 mM ammonium phosphate (monobasic) in 0.1% TFA, a recrystallization step with ethanol/acetone/0.1% TFA (6:3:1; v/v/v) was performed. MALDI-TOF and MALDI-TOF/TOF spectra were acquired using an Ultraflex II TOF/TOF mass spectrometer (Bruker, Germany). The appropriate peptide mixtures were first analyzed in the reflectron mode, followed by TOF/TOF analysis in the LIFT mode. For MS and MS/MS experiments, the mass spectrometer was calibrated externally with a mixture of bradykinin (1-7), angiotensin I and II, substance P, bombesin, renin, ACTH (1-17), ACTH (18-39), somatostatin and oxidized insulin β-chain. The FlexControl 2.4 software controlled the Ultraflex II and the masses (m/z) were annotated using the FlexAnalysis 2.4 software (Bruker, Germany). Labeled TOF/TOF spectra were further processed with the BioTools 3.0 software package using the Mascot MS and MS/MS Ion search option and the RapiDeNovo extension (Bruker, Germany) to obtain de novo sequence information. 2.7. Database Searches. Searching within protein or DNA databases using different search algorithms such as Mascot, Sequest or Pro Found usually enables identification of proteins or homologous ones, if they are already described and the quality of the spectral data is sufficient. For species with genomes not already sequenced, the utilization of such protein or DNA-databases is restricted.31 If the protein identity among different species is less than 70%, the peptide mass fingerprint (PMF) alone is not sufficient for protein identification.32 Identification can then be carried out using correlations of uninterpreted MS/MS peptide spectra with the predicted spectra from peptides of the same mass contained in the databases.33 Cross-species protein identification with uninterpreted MS/MS spectra using Mascot has been demonstrated by Kim et al.34 For unknown proteins or proteins from species with an unsequenced genome, it is necessary to determine partial or complete amino acid sequences from MS/MS spectra using either manual or automated de novo peptide sequence analysis methods.33 The resulting sequence information can then be loaded into database search engines such as MS BLAST and searched against available databases (e.g., NCBInr, SwissProt) for sequence homologies from phylogenetically related species. This sequence similarity-based approach allows the cross-species identification of unknown proteins, that is, less conserved proteins in closely related organisms and conserved proteins in distantly related ones.35 Uninterpreted MS/MS spectra of the tryptic peptides derived from the gel spots were searched against protein sequences from the Swiss-Prot database using the Mascot search program (www.matrixscience.com). At most, one missed cleavage for tryptic peptides was allowed. Carbamidomethylation of cysteine and oxidation of methionine as modifications were selected. The mass tolerances for the precursor ions and the resulting fragments were set to 50 ppm and 0.1-0.2 Da, respectively.
De novo peptide sequences were deduced from the annotated fragment spectra of appropriate precursor ions using RapiDeNovo (precursor mass tolerance adjustment, 50-125 ppm; fragment mass tolerance adjustment, 0.2-0.8 Da; variable modification, oxidation of methionine; fixed modification, carbamidomethylation of cysteine). Homology searches were executed with the MS BLAST search engine (http://genetics. bwh.harvard.edu/msblast) and performed with the following settings: Program, blast2p; Database, Swiss-Prot; Matrix, PAM30MS; Expect, 100; other advanced options, nogap-hspmax100-sort_by_totalscore-span1. The sequences were edited according to the rules described by Shevchenko et al.36 2.8. Glycan Analysis. The haptoglobin was deglycosylated with the enzyme PNGaseF37 from the excised gel spots and in solution from the purified Hp. The enzymatic cleaved glycans derived from the different gel spots were recombined prior to MALDI MS analysis. Permethylation was performed according to Ciucanu38 to achieve better mass signals and stabilization of the sialic acid residues. Briefly, a NaOH/DMSO base and methyliodide (Sigma Aldrich) were added to the sample (dissolved in anhydrous DMSO). The reaction vial was incubated for 10 min. The methylated glycans could then be extracted with dichlormethan (DCM), which was subsequently evaporated under a nitrogen stream. The glycans were redissolved in 20 µL of methanol before application to MALDI analysis. 2,5-Dihydroxybenzoic acid (DHB) was used as a suitable matrix for methyl-esterified oligosaccharides. It consists of 5 g/L DHB in acetonitrile/0.1% TFA (1:2, v/v). Database searches for glycan mass comparison were performed with the database “functional glycomics”: http://functionalglycomics.org/glycomics/.
3. Results and Discussion 3.1. Hp Concentrations in the Plasma of Harbor Seals. In this study, 10 seals were investigated. Among them were four wild-life animals, four seals living in captivity and two fatally ill animals. The Hp concentrations in plasma varied between 0.1 and 2.1 mg/mL. In a study on 71 free-ranging harbor seals of the German and Danish Wadden Sea, Kakuschke found a reference range between 0.2 and 1.6 mg/mL, which is to be considered to be a normal variation.39 The seals under investigation cover animals whose Hp level is within the normal range. The two ill animals had abnormally high levels as expected and one seal had an outstanding low blood concentration level. Elevated Hp levels are indicative of acute phase reactions in response to infection, inflammation, trauma or toxicological damage. This is shown in several investigations. Mellish et al. measured elevated Hp concentrations in Steller sea lions (Eumetopias jubatus) up to 2 weeks after branding and baseline levels after 7-8 weeks.40 Zenteno-Savin et al. have described higher levels of Hp in the plasma of Steller sea lions from areas where the populations are in decline compared to stable populations.41 Hp is significantly reduced during hemolytic episodes, because of the increased removal rate of the Hp-Hb complex as shown in a study on river otters.42 3.2. Isolation of Hp from Plasma Samples. Plasma is defined as the soluble component of the blood. It contains a large variety of proteins, derived from different cells and tissues8 with different chemical and physical features. Furthermore, the protein concentrations lie in a dynamic range covering more than 10 orders of magnitude.7 The highly abundant proteins such as albumin, immunoglobulin, transferrin and lipoproteins interfere with the detection of the less abundant proteome.43 Recently, the use of lectins has increased Journal of Proteome Research • Vol. 8, No. 6, 2009 2925
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Figure 2. Visualization of the separated Hp on a 2-DE derived from a free ranging animal. The characteristic “train of spots” results due to an altered glycosylation. The five main spots labeled A-E of the β-chain can be identified. The R-chain is detected at lower molecular weight.
Figure 1. Flowchart depicting the isolation and characterization process of haptoglobin from the seal (Phoca vitulina). Blood sampling was carried out with EDTA monovettes. After centrifugation, the blood plasma was applied directly to the downstream process, which starts with glycoprotein enrichment due to immobilized lectins. This is followed by SEC and IEX and visualization of the success of the separation by 2-DE. The gel serves as matrix for a 2-fold enzyme application. First, PNGaseF detaches the N-glycans that were permethylated prior to MALDI TOF analysis, and second, proteolytic enzymes (trypsin or GluC) were applied for the amino acid sequence determination.
in popularity for the fractionation of glycoproteins from complex matrices,44,45 because they are even able to enrich less concentrated glycoproteins and separate several highly abundant ones. ConA is a suitable lectin for the enrichment of a relative large amount of glycoproteins. This is necessary for a comprehensive isolation of the different glyco-isoforms of Hp. The lectins specificity is toward mannose and with a lower affinity to glucose. It is noteworthy that mannose does not have to be in a terminal position. ConA is capable to bind the trimannosyl core of diantennary N-glycans. Only higher branched N-glycans that do not possess terminal mannose will be discriminated by the lectin. The optimized complete procedure for Hp isolation is depicted in Figure 1. The blood samples were collected and centrifuged to remove the blood cells. They were then exposed to the affinity adsorbent with immobilized ConA without further preparation or prefractionation. Two following chromatographic steps were sufficient to obtain the Hp in a pure state. The isolated glycoprotein Hp was amenable to enzyme catalysis. First, the glycans had to be detached with PNGaseF and could be analyzed separately. The protein backbone underwent further digestions with proteolytic enzymes for the achievement of peptide sequence information by means of MALDI experiments. The isolated haptoglobin of a healthy animal, whose β-chain presents itself in five different spots (numbered A-E) is presented in Figure 2. Changes in the protein glycosylation 2926
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Figure 3. Comparison of the five mass spectra derived after deglycosylation and tryptic digestion of the β-chain spots (annotated from A to E in Figure 2). The occurrence of the same tryptic peptides proves the identical primary structure of the Hp β-chain.
account for the characteristic “train of spots” generated due to different isoelectric points and molecular weights. The differences in the pI arise from the varying degree of sialylation; differences in the molecular weight originate from a missing
Characterization of Hp in the Blood Plasma of Harbor Seals
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Table 1. Cross-Species Identification of the Seal Homologue of Haptoglobin by MASCOT MS/MS Ion Search and MS BLAST Data Base Search
a
Org., organism; CF, C. familiariz; SS, S. scrofa.
b
sequence from the Swiss-Prot database, ion scores >21 indicate identity or extensive homology (p
3000 Da) may lead to incomplete fragmentation. A second enzyme may fill the gap and provide supplemental information about the primary structure of the protein. The peptide sequences determined from the R- and β-chains obtained from either tryptic or endoproteinase GluC digests can be retraced in the Supporting Information table. The entire amino acid sequence of the seal Hp is summarized in Figure 4 (the lowest line in black letters). The sequence was elucidated to 97%. The black bars under the
Characterization of Hp in the Blood Plasma of Harbor Seals
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Figure 5. Schematic presentation of the seal Hp. Disulfide bonds derived from the homology to dog and human Hp and the potential glycosylation sites deduced from the consensus amino acid sequence Asp-Xxx-Ser/Thr are annotated.
sequence point out the elucidation of the primary structure determined with tryptic peptides and the gray bars indicate GluC peptides. Four small tryptic peptides occur that could not be detected in the MS spectra (amino acids 1-2, 60-64, 176-177 and 245-248). Two of them could be covered with a GluC fragment (amino acid 176-177 and 245-248). Unfortunately, the GluC digest does not provide relevant peptides at the beginning (amino acid 1-2) and for the amino acids 60-64. To demonstrate the close phylogenetic relationship between P. vitulina and C. familiariz, the sequence is pasted in the midline in Figure 4. The sequence homology to P. vitulina is over 89%. Comparison of the P. vitulina and C. familiariz sequence with the human Hp1-1 sequence reveals the location of variable and conserved amino acids. Human Hp has a tetra chain alignment (β-R-R-β) of the subunits, while other mammals possess only R-β conformations and just as in other animals only one subtype of seal Hp exists. The disulfide bond between both R-chains in human Hp is located at Cys15. This position contains no cysteine residue either in seal or dog sequences. All other cysteine residues (marked with frames) responsible for the intramolecular disulfide linkages on the R-chain at positions Cys34, Cys68, Cys72 and Cys189, Cys232, Cys263, Cys274, Cys304 on the β-chain have the same position in the molecule. The spatial vicinity required for the formation of a disulfide bond suggests an identical three-dimensional structure of these proteins. Unfortunately, no data are available for the secondary and tertiary structures of Hp. The seal peptide chain exhibits four possible attachment sites for N-glycans located at Asn12, Asn103, Asn107 and Asn214, which are underlined in gray. The glycosylation site Asn12 (on the R-chain) reveals a distinctive feature. The dog is the only animal with known R-chain glycosylation. Despite the potential glycosylation motif, no glycans could be detected on the seal’s R-chain. This is confirmed by the constitution of the R-chain in a single spot on 2-DE. The consensus recognition sequence Asn-Xxx-Ser/Thr is in fact necessary for N-linked glycosylation but not implicitly sufficient. Many possible glycosylation sites are not glycosylated and little is known about the protein signals that control the efficiency of the oligosaccharide precursor transfer. The β-chain glycosylation is more complex. The remaining three glycosylation sites are responsible for the typical spotting pattern in the gel. The glycosylation site at Asn107 belongs to the conserved regions and is therefore assumed to be important for the molecules function and/or folding. All other motifs show variable conservation and are hence unlikely to affect the overall protein folding.
Figure 6. Characteristic haptoglobin glycoprofiles: (a) derived from diseased animals, (b) free ranging animals, and (c) seals living in captivity. All profiles are dominated by a disialylated biantennary complex type N-glycan with a permethylated weight of 2793 g/mol (2224.01 g/mol native weight). Glycans reduced by a single NeuAc and further by a galactose residue are always present in the profiles. Differences arise from the smaller and the fucosylated glycans, which may act as potential biomarkers. (*) Structures were annotated due to their molecular weight. Other structures were confirmed by MS/MS experiments.
Two of them are in near vicinity (Asn103 and Asn107), which could be considered to be affected by steric hindrances for glycan attachment. Investigations on the glycosylation efficiency were done with respect to the influence of the Xxx amino acid on the consensus sequence, because it affects the precursor transfer. It is known that the presence of proline completely blocks glycosylation. The first binding motif on the β-chain contains Gly in the Xxx position, which is associated with highly efficient glycosylation up to 100%,48 while the following glycosylation site (Asn107) possesses Leu which contains a large hydrophobic side chain and in general impairs glycosylation efficiency. On the other hand, this site belongs to the conserved regions of Hp. Hp from dogs and humans Hp exhibits this glycosylation site in the same position; hence, Journal of Proteome Research • Vol. 8, No. 6, 2009 2929
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Figure 7. MS/MS fragmentation pattern of the disialylated biantennary complex type N-glycan with a permethylated weight of 2793 g/mol. The benefit of the permethylation is the stabilization of the sialic acid residues and better fragmentation characteristics. The cleavage of the glycosidic bonds allows the unequivocal classification of the N-glycans.
it is assumed to carry an oligosaccharide chain. The last motif at Asn214 possesses a Tyr side chain, which results in a 75% glycosylation efficiency, as determined with site directed mutagenesis by Shakin-Eshleman et al.48 The missing glycosylation at this position is thought to be responsible for the mass shift in the Hp β-chain, observable in the 2D gel. Furthermore, the Hp assay that requires binding to Hb reveals no differences for the haptoglobins from different species, suggesting that the variance in glycosylation site conservation does not influence Hb binding. Figure 5 provides a schematic presentation of the R-β alignment of the molecule including the disulfide bonds and glycosylation sites. 3.4. Characterization of the N-Glycans. The unique principle of the biosynthesis of glycoproteins, including the transfer of an oligosaccharide precursor to the nascent peptide chain with subsequent trimming and transfer of monosaccharides, gives rise to branched oligosaccharides which constitute a specific trimannosyl core. The varying terminations of the branches make them classifiable into “high-mannose”, “complex” and “hybrid” glycans. The mammalian N-glycans are remarkably well-conserved, which facilitates the glycan analysis in such a way, that for a given mass of a glycan the monosaccharide composition can be calculated and thus a certain structure is likely to occur. Difficulties raises from the natural microheterogeneity of the attached glycans that complicates the determination of the intrinsic glycosylation of the molecules investigated. The concept of “biochemical individuality” has to be taken into consideration, that is, every living organism in its environment represents a biochemically unique system. First of all, it is desirable to assess the state of health or “normalcy” within the organisms under study, which is not trivial, because healthy animals underlie a natural variability. Further on, the number of individuals and their glyco-profiles have to be judged on a statistical basis to distinguish between the “apparently healthy” and “diseased” individuals. This is particularly complicated, when working on free living animals, where the samples are scarce and the diagnosis of these animals is difficult. To improve the reproducibility of the glyco-profile analysis, the cleaved oligosaccharides were permethylated prior to MS 2930
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Rosenfeld et al. analysis. This led to significant analytical benefits, for example, better ionization and fragmentation as well as stabilization of the sialylated (acidic) glycans. Typical glycoprofiles derived from 3 seals of different origins are displayed in Figure 6. The upper profile is derived from a diseased animal, the profile in the middle originates from a free-ranging animal, and the profile at the bottom was obtained from a seal living in the seal station Friedrichskoog. All profiles, irrespective of the animals origin and health status, are dominated by the m/z 2793 disialylated diantennary complex type glycan and this turned out to be in good reproducibility. Three analytical replicates were accomplished for each animal (provided in the Supporting Information). The not permethylated weight of the dominating glycan is 2224.01 g/mol. According to the work of He et al., this glycan is also the most abundant in human haptoglobin, but they also found more complex glycan structures composed of tri- and tetraantennary oligosaccharides.21 Nakano et al. have also detected such tri- and tetraantennary glycans in human Hp and correlated the degree of fucosylation of these glycans with cancer diseases.49 The seal pattern exhibits moreover the monosialylated oligosaccharide at 2431 m/z (native weight is 1932.75 g/mol). The lack of NeuAc brings galactose into the terminal position. If the galactose residue is missing, the weight of the glycan reduces to 2227 m/z (1770.61 g/mol). These glycans are always present in the haptoglobin glycoprofiles. In fact, the number of animals investigated was insufficient to interpret differences in the glycoprofiles. However, the direction for ongoing investigations could be identified. The degree of fucosylation could also be observed in order to find a possible biomarker in the future. Okuyama et al. have reported an elevated degree of fucosylated Hp in the case of pancreatic cancer.50 The appearance of fucosylated Hp has further been investigated for other diseases such as colon cancer, hepatocellular carcinoma, liver cirrhosis, and gastric cancer. These Hp isoforms are not only indicative of liver diseases, even though Hp is mainly produced in the liver. Okuyama et al. has further proposed a possible mechanism for fucosylation.50 The MS/MS fragmentation pattern of the 2793 m/z precursor is exemplified in Figure 7. Further MS/MS experiments of the other glycans are outlined in the Supporting Information. The fragmentations of the oligosaccharides confirm the specified structures. In general, the [M + Na]+ parent ion is produced by the DHB matrix. For the fragmentation of this parent ion, three types of cleavages are common: loss of the adduct; glycosidic cleavage resulting from the breaking of a bond linking two sugar rings, which can occur at two or more sites in different parts of the molecule; and cross-ring cleavages that involve the breaking of two bonds.51 The most informative cleavage is that of the glycosidic bond. The fragment spectrum reveals the successive additions of monosaccharides that build up the entire oligosaccharide. For more details on the different mechanisms of saccharide fragmentation, refer to Harvey.52
4. Concluding Remarks The isolation and structural elucidation of seal Hp is described in this work. The protein sequence was determined to 97%. The glycans were analyzed after permethylation. The approach is to investigate animals derived from three different origins (free ranging animals, living in captivity and fatally ill). At this stage, however, this study does not allow haptoglobin to be defined unambiguously as a glyco-biomarker, because of the limited number of animals that have been investigated
Characterization of Hp in the Blood Plasma of Harbor Seals to date. The future direction is an extended screening of normal and diseased seals along with experiments to observe whether it is the core fucosylation of serum Hp that might be decreasing in diseased seals. The work presented here is the first step to augment these proteomic approaches by glycomic investigations and may form the basis for the development of a diagnostic tool for the monitoring of the health of the harbor seal population in the North Sea. It is a part of comprehensive investigations on marine mammal health that includes the influence of pollutants such as heavy metals and organic contaminants on the immune status of these animals. A better understanding of the interactions between toxic chemical and environmental factors is a fundamental requirement for the efficient management and protection of aquatic systems. Abbreviations: APP, acute phase protein; ConA, lectin concanavalin A; Hb, hemoglobin; Hp, haptoglobin.
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