Liver membrane proteome glycosylation changes in mice bearing an extra-hepatic tumor

Abstract

Cancer is well known to be associated with alterations in membrane protein glycosylation (Bird, N. C., Mangnall, D., and Majeed, A. W. (2006) Biology of colorectal liver metastases: A review. J. Surg. Oncol. 94, 68-80; Dimitroff, C. J., Pera, P., Dall'Olio, F., Matta, K. L., Chandrasekaran, E. V., Lau, J. T., and Bernacki, R. J. (1999) Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem. Biophys. Res. Commun. 256, 631-636; and Arcinas, A., Yen, T. Y., Kebebew, E., and Macher, B. A. (2009) Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns. J. Proteome Res. 8, 3958-3968). Equally, it has been well established that tumor-associated inflammation through the release of pro-inflammatory cytokines is a common cause of reduced hepatic drug metabolism and increased toxicity in advanced cancer patients being treated with cytotoxic chemotherapies. However, little is known about the impact of bearing a tumor (and downstream effects like inflammation) on liver membrane protein glycosylation. In this study, proteomic and glycomic analyses were used in combination to determine whether liver membrane protein glycosylation was affected in mice bearing the Engelbreth-Holm Swarm sarcoma. Peptide IPG-IEF and label-free quantitation determined that many enzymes involved in the protein glycosylation pathway specifically; mannosidases (Man1a-I, Man1b-I and Man2a-I), mannoside N-acetylglucosaminyltransferases (Mgat-I and Mgat-II), galactosyltransferases (B3GalT-VII, B4GalT-I, B4GalT-III, C1GalT-I, C1GalT-II, and GalNT-I), and sialyltransferases (ST3Gal-I, ST6Gal-I, and ST6GalNAc-VI) were up-regulated in all livers of tumor-bearing mice (n = 3) compared with nontumor bearing controls (n = 3). In addition, many cell surface lectins: Sialoadhesin-1 (Siglec-1), C-type lectin family 4f (Kupffer cell receptor), and Galactose-binding lectin 9 (Galectin-9) were determined to be up-regulated in the liver of tumor-bearing compared with control mice. Global glycan analysis identified seven N-glycans and two O-glycans that had changed on the liver membrane proteins derived from tumor-bearing mice. Interestingly, α (2,3) sialic acid was found to be up-regulated on the liver membrane of tumor-bearing mice, which reflected the increased expression of its associated sialyltransferase and lectin receptor (siglec-1). The overall increased sialylation on the liver membrane of Engelbreth-Holm Swarm bearing mice correlates with the increased expression of their associated glycosyltransferases and suggests that glycosylation of proteins in the liver plays a role in tumor-induced liver inflammation.

Fig. 1.

Relative abundance of glycosyltransferases and mannosidases determined by peptide IPG-IEF followed by label-free quantitation. NSAF values describe the relative fold difference among samples with p ≤ 0.05. *Selected glycosyltransferases that did not change in abundance (supplemental Table S1).

Fig. 2.

Cell surface lectins play a role in the immune system and were found to be significantly up-regulated in the liver of tumor-bearing mice p ≤ 0.05. A detailed glycan array profiling of the Kupffer cell receptor has indicated it is primarily a galactose- and GalNAc-binding receptor (74).

Fig. 3.

Combined LC-ESI-MS (retention time 20–65 min) of globally released N-linked oligosaccharides from mouse liver membrane proteins from control (A) and tumor-bearing mice (B). Combined extracted ion chromatogram (EIC) of m/z (941.3)²⁻, (1022.4)²⁻, and (1038.9)²⁻ and average MS (retention time 21–28 min) of control (C) and tumor-bearing mice (D). Ions corresponding to m/z (1022.4)²⁻ and (1038.9)²⁻ were observed in low abundance in the average MS spectra in both control (A) and tumor-bearing (B) mice but were clearly observable by their combined EIC (C and D). Averaging MS spectra of the narrower retention time (21–28 min) reveals the presence of m/z (1022.4)²⁻. Some major ions corresponding to N-glycans have been assigned possible structures (supplemental Table S2). *Representative [M-2H]²⁻ ions correspond to identified structures found only in tumor-bearing mice (Table I).

Fig. 4.

Extracted ion chromatogram (EIC) of m/z (1127. 4)²⁻ shows the different ratios of the biantennary N-glycan with two NeuGc attached shown in (A) control (n = 3, p ≤ 0.05) and (B) tumor-bearing mouse liver membranes (n = 3, p ≤ 0.05) (C) MS/MS fragmentation of glycan isomer observed at 41 min in tumor-bearing mice indicates the position of the two NeuGc. Isomer 1 and 2 are two isomeric forms of m/z (1127.4)²⁻.

Fig. 5.

N-linked oligosaccharides were treated with α(2,3) neuraminidase to remove α(2,3) linked NeuGc on di-sialylated biantennary N-glycan corresponding to m/z of (1127. 4)². EIC of m/z (1127.4)²⁻ with no neuraminidase digestion and combined MS (A) over 40–65 min. EIC of m/z (1127.4)²⁻ after a overnight α(2,3) neuraminidase digestion of N-linked oligosaccharides and combined MS (B) over 40–65 min. * Generated additional di-sialylated N-glycans.

Fig. 6.

Identification of N-glycan with three glycolyl neuraminic acids. Structures are depicted as biantennary N-glycans because this is the most likely configuration of the compositions in the mouse. EIC of (1127.4)²⁻ and (1280.9)²⁻ and combined MS spectrum (A) over 40–65 min. Digestion of purified N-linked oligosaccharides with α(2,3,6) neuraminidase for 10 min and combined MS spectrum (B) over 40–65 min. Overnight digestion of purified N-linked oligosaccharides with α(2,3,6) neuraminidase and combined MS spectrum (C) over 40–65 min (D). MS/MS fragmentation of the resultant monosialylated N-glycan (m/z of (973.8)²⁻) confirms a NeuGc attached to a GlcNAc(m/z ion 509.1) thus predicting the structures shown.

Fig. 7.

Combined EIC of (675. 3)¹⁻, (691.3)¹⁻, (966.2)¹⁻, (982.3)¹⁻, (998.4)¹⁻, and (681.3)²⁻ and average MS of O-linked oligosaccharides (retention time 17.2–17.9 min) from liver membranes from control (A) and tumor-bearing mice (B) MS/MS fragmentation of m/z (998.4)¹⁻ (C) and MS/MS fragmentation of m/z (681.3)²⁻(D) confirming the structures shown.

Fig. 8.

1D SDS-PAGE followed by in-gel PNGase F digestion of proteins of decreasing mass to release N-linked oligosaccharides. Combined MS of 20–65min of the N-glycans released from the proteins with each band show a similar global profile to the total protein glycoprofile.