Biomed Res Int. 2013;2013:834790. doi: 10.1155/2013/834790.

Development of a novel system for mass spectrometric analysis of cancer-associated fucosylation in plasma α1-acid glycoprotein.

Takayuki Asao,1 Shin Yazawa,1,2 Toyo Nishimura,2 Takashi Hayashi,3 Hideyuki Shimaoka,4 Abby R. Saniabadi,5 and Hiroyuki Kuwano1

1Department of General Surgical Science, Gunma University Graduate School of Medicine, Maebashi 371-8511,  Japan
2Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0192,  Japan
3Institute of Biomedical Innovation, Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0192,  Japan
4S-BIO Business Division, Sumitomo Bakelite Co., Ltd., Tokyo 140-0002,  Japan
5JIMRO Co., Ltd., Tokyo 151-0063,  Japan

 

Abstract

Human plasma α1-acid glycoprotein (AGP) from cancer patients and healthy volunteers was purified by sequential application of ion-exchange columns, and N-linked glycans enzymatically released from AGP were labeled and applied to a mass spectrometer. Additionally, a novel software system for use in combination with a mass spectrometer to determine N-linked glycans in AGP was developed. A database with 607 glycans including 453 different glycan structures that were theoretically predicted to be present in AGP was prepared for designing the software called AGPAS. This AGPAS was applied to determine relative abundance of each glycan in the AGP molecules based on mass spectra. It was found that the relative abundance of fucosylated glycans in tri- and tetra-antennary structures (FUCAGP) was significantly higher in cancer patients as compared with the healthy group (P < 0.001). Furthermore, extremely elevated levels of FUCAGP were found specifically in patients with a poor prognosis but not in patients with a good prognosis. In conclusion, the present software system allowed rapid determination of the primary structures of AGP glycans. The fucosylated glycans as novel tumor markers have clinical relevance in the diagnosis and assessment of cancer progression as well as patient prognosis.

PMID: 23509786

http://dx.doi.org/10.1155/2013/834790

 

SUPPLEMENT

Human α1-acid glycoprotein (AGP) is a major plasma glycoprotein with a molecular weight of 41- 43 KDa. As illustrated in Figure 1, AGP possesses five complex N-linked glycans per molecule attached at positions 15, 38, 54, 75 and 85 Asn, and consisted of di-, tri- and tetraantennary glycans. Each glycan chain is commonly sialylated through α2,3 and/or α2,6 linkages at the terminal [Gal] residues giving rise to a very low pI of the molecule, and some tri- and tetraantennary glycan chains possess [Fuc] residues attached to the penultimate [GlcNAc] residues through α1,3 linkages forming Lex (Galβ1,4[Fucα1,3]GlcNAc) or sialyl Lex (NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAc) structure, which is only type of fucosylation being expressed on AGP. It has frequently been observed that glycosylation occurs in each antenna with considerable heterogeneity and that the tetraantennary glycans elongate with repeating lactosamine structures, (Galβ1,4GlcNAcβ)n in advance of α2,3/α2,6 sialylation at the terminal [Gal] residues.

Recently, with the aid of a crossed affinoimmunoelectrophoresis (CAIE), glycoforms of AGP were characterized by using Con A lectin, Aleuria aurantia lectin (AAL) and anti-human AGP antibody in cancer patients and then were observed over a long time period.1 It was found that patients with advanced malignancies who had AGP glycoforms containing highly fucosylated branched glycans for long periods after surgery had a poor prognosis, while patients without such glycoforms appeared to have a good prognosis irrespective of their clinical stages. The CAIE method has an advantage to analyze AGP glycoforms without purification of AGP but is not applicable for a large number of samples in a short period of time.

In this study, we developed a rapid assessment method for primary structures of AGP glycans by means of a mass spectrometer. Establishment of a simple procedure for purification of plasma AGP and operation software called AGPAS represented a novel advance for mass spectrometric analysis of AGP glycans from a large number of clinical samples. For identification of individual glycans that were predicted to be present in AGP, the glycan molecules were abbreviated by using a four-digit number indicating the number of [Gal], [GlcNAc], [Fuc] and [NeuAc] residues of each glycan except the common core structure ([Man]3[GlcNAc]2) (Figure 1). Accordingly, 453 theoretically predicted N-glycans were determined from 4193 glycans extracted from the GlycMod Tool available through the ExPASY.2 In addition, 154 methylated glycans with maximum probability were predicted to yield from methylesterification of [NeuAc] residues attached to the terminal [Gal] residues through the labeling process, and finally, 607 glycans were extracted in total for constituting the database of the AGPAS (Figure 2). This system allowed us not only to identify individual glycans but also determine their relative abundance within a short period of time. Therefore, the relative abundance of α1,3fucosylated tri- and tetraantennary glycans in AGP (FUCAGP) was found to be significantly higher in cancer patients than in healthy controls. Furthermore, increased FUCAGP was observed specifically in patients with a poor prognosis (Figure 3), which was consistent with our previous analyses of AGP glycoforms in large numbers of cancer patients together with their following-up studies.1 The methods applied in this study should be appropriate for processing large numbers of plasma samples from cancer patients to determine a novel biomarker in AGP glycans.

 

REFERENCES:

  1. Hashimoto S, Asao T, Takahashi J, Yagihashi Y, Nishimura T, Saniabadi AR, Poland DCW, van Dijik W, Kuwano H, Kochibe N, Yazawa S. 2004. α1-Acid glycoprotein fucosylation as a maker of carcinoma progression and prognosis. Cancer 101:2825-36.
  2. http://web.expasy.org/glycomod/

 

LEGENDS FOR FIGURES

World Biomedical Frontiers Fig 1Figure 1.  Examples of differently branched N-linked glycans in AGP and their abbreviated notations used with a four-digit number in AGPAS. GlcNAc, N-acetyl-D-glucosamine; Man, D-mannose; Gal, D-galactose; NeuAc, N-acetylneuraminic acid (sialic acid); Fuc, L-fucose; Asn, asparagine.

 

World Biomedical Frontiers Fig.2Figure 2.  Establishment of operation software, AGPAS to assist analysis of N-glycans in AGP. Six hundred and seven glycans were summed up in total for constituting the database of the AGPAS through three steps of the extraction.

World Biomedical Frontiers Fig 3Figure 3.  Changes in relative abundance of bi-, tri- and tetraantennary glycans and its fucosylated glycans in AGP isolated from followed up patients with a good (A) and a poor (B) prognosis. Patients were from gastric cancer with stage IIA (A) and with stage IIIB (B), respectively. The patient B died at 231 POD due to disease recurrence. Relative abundance of bi-antennary glycans and α1,3fucosylated-(tri- + tetraantennary) glycans (FUCAGP), was indicated alone at each POD. Relative abundance of tri- and tetraantennary glycans was indicated together with that of its fucosylated ones at the same POD. POD, postoperative days.

Contact:

Shin Yazawa, Ph.D.

Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima 771-0192, Japan.

Depet. General Surgical Science, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan

syazawa@titan.ocn.ne.jp

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