Increased Outer Arm and Core Fucose Residues on the N-Glycans of

Article pubs.acs.org/jpr

Increased Outer Arm and Core Fucose Residues on the N‑Glycans of Mutated Alpha‑1 Antitrypsin Protein from Alpha‑1 Antitrypsin Deficient Individuals Cormac McCarthy,† Radka Saldova,‡ M. Emmet O’Brien,† David A. Bergin,† Tomás P. Carroll,# Joanne Keenan,§ Paula Meleady,§ Michael Henry,§ Martin Clynes,§ Pauline M. Rudd,‡ Emer P. Reeves,*,† and Noel G. McElvaney† †

Respiratory Research Division, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, University College Dublin, Dublin 4, Ireland # Alpha One Foundation, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland § National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland ‡

S Supporting Information *

ABSTRACT: Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease−antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1−3) (p = 0.04) and core (α1−6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention. KEYWORDS: N-linked glycans, alpha-1 antitrypsin, alpha-1 antitrypsin deficiency, emphysematous lung disease, fucosylation, sialyl Lewis X, plasma

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INTRODUCTION Alpha-1 antitrypsin (AAT) is a 52 kDa glycoprotein found circulating in human plasma at a concentration of 1−2g/L.1 AAT is a serine protease inhibitor encoded by the SERPINA1 gene on 14q32.1. It is synthesized predominantly within hepatocytes,2 but other cells such as epithelial cells,3 monocytes,4 macrophages and neutrophils,5,6 intestinal epithelial cells7 and cancer cells have also been shown to produce smaller quantities of the protein.8 The AAT molecule consists of a polypeptide chain composed of 394 amino acids and is post-translationally modified by glycosylation in the endoplasmic reticulum. N-Glycosidically linked oligosaccharides are added at three asparagine residues at 46, 83, and 247 on the peptide,9−11 and these carbohydrate residues contribute 12.5% of the molecular weight of AAT. AAT plays a pivotal role in the lung by averting destruction of alveolar structures and connective tissue breakdown which © 2013 American Chemical Society

results in emphysema. AAT primarily acts as an inhibitor of serine proteases derived from degranulating neutrophils including neutrophil elastase (NE), cathepsin G and proteinase 3.12 Structurally AAT contains nine α-helices, three β-sheets and a reactive center loop (RCL) with an active methionine residue at position 358. AAT inhibition of serine proteases utilizes a pseudo substrate consisting of 20 amino acids that are recognized by the target protease.13,14 When NE interacts and cleaves this residue, it induces an irreversible conformational change in AAT which inactivates and incorporates NE into the AAT molecule.15 Moreover, AAT is an acute phase protein16 with plasma concentrations increasing 2- to 4-fold17 within hours of acute inflammation or infection.18 Increasingly, it has been recognized that AAT possesses additional anti-inflammaReceived: July 19, 2013 Published: December 4, 2013 596

dx.doi.org/10.1021/pr400752t | J. Proteome Res. 2014, 13, 596−605

Journal of Proteome Research

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tory properties beyond antiprotease activity including attenuation of oxidative stress,19 regulation of leucocyte migration,4,20 inhibition of apoptosis,19,21 binding to pro-inflammatory cytokines,5 and an involvement in tissue repair mechanisms.22 These unique anti-inflammatory properties affect several cell types and modulate inflammation caused by both host and microbial factors. Alpha-1 antitrypsin deficiency (AATD) is an autosomal codominant condition affecting an estimated 3.4 million individuals worldwide.23 The most common form of severe AATD is the PiZZ phenotype, which is caused by a glutamic acid to lysine substitution at residue 342 in the peptide chain. This single amino acid substitution leads to an alteration in the secondary structure of the AAT molecule, which in turn leads to aberrant protein folding and accumulation of misfolded ZAAT in the ER of hepatocytes and other AAT-producing cells.24,25 AATD patients have serum AAT levels approximately 10% that of circulating AAT found in healthy individuals (PiMM) and consequently have reduced NE inhibitory capacity and are susceptible to severe lung disease.26 A number of different glycoforms of AAT have been known to exist since the mid-1970s,27 and the glycoform pattern upon isoelectric focusing is routinely used to phenotype AATD individuals. Despite the observation of glycoform variants in AATD, very little study regarding the role that glycosylation plays in AATD has been carried out to date. This renewed interest in AAT glycosylation follows evidence of improved stability, increased serum half-life and functional efficacy of treatment with glycosylated AAT for augmentation therapy in AATD.9,28 The glycans of the PiMM protein (M-AAT) have been identified and studied, with 9 different glycoforms of M known, often classified as M0 to M8.11 Multiple glycoforms of Z-AAT have been reported and visualized on 2-dimensional gel electrophoresis, with 6 glycoforms previously reported;10,11 however, no glycoanalysis has been published on Z-AAT. Although glycans do not have a role in the antiprotease function of M-AAT, glycosylation plays a key role in augmenting the anti-inflammatory effect of M-AAT.5,29 In this regard, it has been demonstrated that glycosylated M-AAT exerts a greater immunomodulatory effect upon IL-8 compared to nonglycosylated AAT.5 In this latter study, the inhibitory effect of glycosylated M-AAT on neutrophil IL-8 cell responsiveness and chemoattractant-induced cytoskeletal rearrangements including polymerization of globular actin was demonstrated. The observed reduction in chemotaxis by AAT involved direct binding of glycosylated AAT to IL-8 thereby preventing CXCR1 engagement and was alleviated by increasing the concentration of chemoattractant, suggesting that this inhibitory effect may be overcome in the presence of sufficient chemokine.5 Moreover, in a number of inflammatory and malignant conditions, the glycosylation profile of AAT changes, highlighting the importance of glycans in contributing to the immune regulatory function of AAT, independent of antiprotease activity. Thus, we hypothesized that the glycosylation of Z-AAT may differ from that of M-AAT. Hence, the aim of this study was to perform comprehensive glycoanalysis of the Z-AAT protein and identify variance in glycosylation from M-AAT. As glycosylation of AAT can mediate a spectrum of anti-inflammatory effects, this information would lead to a deeper understanding of AATD related disease and impact on future treatment options.

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MATERIALS AND METHODS

Chemicals and Reagents

All chemicals and reagents were of the highest purity and endotoxin free and were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Plasma Samples

The National Alpha-1 Antitrypsin Deficiency Targeted Detection Programme, base at Beaumont Hospital, Ireland, screens individuals with obstructive lung disease and family members of those identified with AATD. Plasma was collected from 5 individuals with a known phenotype of PiZZ AATD (mean age was 46.2 years (range 20−59 years)). All 5 were nonsmoking males with a forced expiratory volume in 1 s (FEV1) greater than 80% predicted and an FEV1/FVC (forced vital capacity) ratio of more than 70%. All individuals were well at the time of blood sample acquisition, with no evidence of acute infection or history of exacerbation in the preceding 6 months. The mean concentration of circulating AAT in the PiZZ individuals was 4.2 μM (range 3.1−5.1 μM). AAT quantification was performed using immune turbidimetry (Olympus AU5400). Control male volunteers (n = 5, mean age 32.2 years (range 23−35 years)) showed no evidence of any disease and had no respiratory symptoms; none were taking medication, all nonsmokers and all proven PiMM phenotype with serum AAT concentrations within the normal range (1−2 g/L). The mean concentration of AAT in the PiMM individuals was 23 μM (range 20.6−26.68 μM). Ethical approval for this study was granted by the Beaumont Hospital Medical Ethics Research Committee. Blood was collected in Sarstedt Monovette tubes containing lithium−heparin. Plasma was immediately isolated by centrifugation of the blood (1000g, 10 min at room temperature) which was then aliquoted and stored at −80 °C until required. A Sebia isoelectrofocusing kit was employed for AAT phenotyping with the HYDRASYS system as previously described.30 Purification of Alpha-1 Antitrypsin from Plasma

AAT was purified from plasma of all 10 individuals (5 ZZAATD individuals and 5 healthy controls) using Alpha-1 Antitrypsin Select (a packed-bed affinity chromatography medium with high selectivity for AAT; GE Healthcare, U.K., Ltd.). Binding Buffer (1 mL of 20 mM Tris, 150 mM NaCl, pH 7.4) was added to 100 μL of AAT-Select Resin which was then centrifuged (10 600g for 5 min at 4 °C) to pellet the resin. The supernatant was removed and the wash step was repeated four times. A volume of 200 μL of plasma from each individual was diluted to 1 mL with binding buffer and then added to prewashed AAT-Select Resin and mixed end-over-end for 1 h at room temperature. The mixture was then centrifuged (10 600g for 5 min at 4 °C) and the supernatant removed. The resin was then washed 10 times in binding buffer, as already described, and bound AAT was eluted from the resin with Binding Buffer containing 2 M MgCl2. AAT was desalted into phosphate buffered saline solution (PBS, pH 7.4) using a Nap-5 Column (GE Healthcare). Protein quantification was performed using BCA Protein Assay Kit (Thermo Scientific, Pierce) and purified AAT (Athens Research) was used to establish a standard curve. The purity of AAT was assessed by SDS-PAGE and Western blot analysis using polyclonal goat antibody against human AAT (0.5 μg/mL, Abcam, Cambridge, U.K.) (Supporting Information Figure S1). 597



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Fluorescent Labeling of Samples

threshold score of 40 and MASCOT significance threshold of 0.05.

2D-Fluorescence Difference Gel Electrophoresis (2D-DIGE) was performed using CyDye (Amersham, GE Healthcare, U.K.) to fluorescently label AAT. Cy2 (yellow), Cy3 (red) and Cy5 (blue) were employed. Cy3 and Cy5 were each added to 6 μg of M-AAT and Z-AAT, respectively. Cy2 was added to a standard pooled sample, composed of 0.5 μg of each M-AAT and Z-AAT sample, totalling 6 μg. For all CyDyes used, a volume of 0.5 μL of 0.4 mM concentration was used per 6 μg of AAT in a total volume of 50 μL. Following CyDye labeling, samples were left on ice in darkness for 30 min; subsequently, samples were pooled and loaded onto immobilized pH gradient (IPG) strips for separation by isoelectric focusing (IEF). Following second dimension SDS-PAGE (12.5% (w/v)), gels were scanned using the Typhoon 9400 variable mode imager (GE Healthcare). Image analysis was performed using the Decyder Software version 6.2 (GE Healthcare).

Removal of N-Linked Glycans from AAT 2-DE Isoforms Using PNGaseF

All AAT glycoforms were cut out of the 2D-PAGE gels. In the 5 PiMM individuals, 4 of the individuals had 7 glycoforms present and one individual had 9 glycoforms present. This totalled 37 glycoforms of M-AAT for analysis. In the 5 PiZZ individuals, 3 individuals had 6 glycoforms present and 2 individuals had evidence of 7 glycoforms seen on 2D-PAGE; hence, a total of 32 glycoforms of Z-AAT were analyzed. Each spot, corresponding to a single glycoform, identified on Coomassie stained gels, was cut into 1 mm2 pieces. N-Glycans were released from the AAT glycoforms and cut from 2DPAGE gels using the high-throughput method described by Royle et al.31 The N-linked glycans were released using peptide N-glycanase F (EC 3.5.1.52, Prozyme, cat number PZGKE5006D) as described previously.32 Briefly, 50 μL of 100 mU/ mL PNGaseF in 20 mM NaHCO3 buffer was added to each gel piece which was left to soak for 5 min and then further covered with 50 μL of 20 mM NaHCO3 buffer. Finally, samples were sealed and incubated at 37 °C overnight.

Separation of Glycoforms by 2-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

The first dimension IEF separation was performed using IPG strips (Amersham, GE Healthcare, U.K.) with a nonlinear (NL) pH gradient of 3−5.6. IEF was performed employing purified AAT (6 μg) in a final volume of 250 μL of IEF Buffer (8 M urea, 1% (v/v) Triton X-100, 4% (w/v) Chaps, 10 mM TrisHCl, 2 M thiourea, dH20) with 0.8% (v/v) ampholytes (IPG 3−5.6 NL) and 65 mM DTT. Separation was carried out using 13 cm IPG strips and an Ettan IPGphor II IEF system (Amersham Bioscience). Strips were separated in increasing voltage from 50 to 8000 V over a 22 h period at room temperature. Following the IEF separation, strips were equilibrated twice for 30 min each time in equilibration buffer (6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mM TrisHCL, pH 6.8). The first equilibration step was carried out in 10 mL of equilibration buffer containing 2% (w/v) DTT and the second equilibration step contained 2.5% (w/v) iodoacetamide. The IPG strips were blotted to remove excess liquid, then were quickly applied to the top of 12.5% (w/v) SDS-PAGE gels and were sealed with agarose sealing solution (1× SDS-PAGE Running Buffer, 0.5% (w/v) agarose and 0.002% (w/v) bromophenol blue). Gels were run overnight at 30 V. Gels were visualized with either Coomassie Blue stain or with the use of fluorescent CyDye imaging. The Coomassie blue stain used for these experiments was 10% acetic acid, 45% methanol, 45% H2O, and 0.2% (w/v) Coomassie Brilliant Blue R250. All M-AAT and Z-AAT gels were stained using the same method.

2-Aminobenzamide (2-AB) Labeling of Glycans

Glycans were fluorescently labeled with 2-aminobenzamide (2AB) by reductive amination.33 Excess 2AB reagent was removed on Whatman 3MM paper (Clifton, NJ, USA) in acetonitrile. Ultra Performance Liquid Chromatography (UPLC)

UPLC was performed using a BEH Glycan 1.7 μm, 2.1 × 150 mm column (Waters, Milford, MA, USA) on an Acquity UPLC (Waters, Milford, MA) equipped with a Waters temperature control module and a Waters Aquity fluorescence detector. Solvent A was 50 mM formic acid adjusted to pH 4.4 with ammonia solution. Solvent B was acetonitrile. The column temperature was set to 30 °C. The following conditions were used: samples were injected in 70% (v/v) acetonitrile, a linear gradient of 30−47% A at 0.56 mL/min in 23 min was employed. Fluorescence was measured at 420 nm with excitation at 330 nm. The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to create a dextran ladder as described previously.34 Exoglycosidase Digestions

All enzymes were purchased from Prozyme (San Leandro, CA, USA) or New England Biolabs (Hitchin, Herts, U.K.). The 2AB-labeled glycans were digested in a volume of 10 μL for 18 h at 37 °C in 50 mM sodium acetate buffer, pH 5.5 (except in the case of jack bean α-mannosidase (JBM) where the buffer was 100 mM sodium acetate, 2 mM Zn2+, pH 5.0), using arrays of the following enzymes: Arthrobacter ureafaciens sialidase (ABS, EC 3.2.1.18), 1 U/mL; Streptococcus pneumoniae sialidase (NAN1, EC 3.2.1.18), 1 U/mL; bovine testes β-galactosidase (BTG, EC 3.2.1.23), 1 U/mL; bovine kidney alpha-fucosidase (BKF, EC 3.2.1.51), 1 U/mL; β-N-acetylglucosaminidase cloned from S. pneumonia, expressed in Escherichia coli (GUH, EC 3.2.1.30), 4 U/mL; jack bean α-mannosidase (JBM, EC 3.2.1.24), 50 U/mL; almond meal alpha-fucosidase (AMF, EC 3.2.1.111), 3 mU/mL. After incubation, enzymes were removed by filtration through a 10 kDa protein-binding EZ filter (Millipore Corporation).34 N-Glycans were then analyzed by UPLC.

Identification of AAT by In-Gel Digestion and Mass Spectrometry Analysis

LC−MS/MS was performed on all individual gel pieces. For detailed methods of LC−MS/MS see Supporting Information. Protein identification was carried out using Proteome Discoverer Software (v1.4) (Thermo Fisher Scientific) using a complementary two stream algorithm search with SEQUEST and MASCOT against a human subset from the UniProtKB/ SwissProt database (downloaded on January 2013) containing 22 991 proteins entries. Carbamidomethylation of cysteine residues was selected as a fixed modification and oxidation of methionine was considered as a variable modification also allowing for two missed cleavages. The following SEQUEST filters were applied: for charge states 1, Xcorr >1.9; 2, Xcorr >2.2; 3, Xcorr >3.75 and Peptide Delta CN (Maximum delta Cn:0.1). The following MASCOT filters were also applied: MASCOT 598



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Statistical Analysis

The D’Agostino & Pearson omnibus normality test was used to asses if the data was normally distributed. The data was not normally distributed; hence, the Mann−Whitney test was employed to compare the peak areas of chromatograms in identifying differences in glycans between M-AAT and Z-AAT. All statistical analysis was carried out using SPSS ver.18 and GraphPad Prism 4.

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RESULTS

The Cathodal Shift and Multiple Glycoforms of Z-AAT

M- and Z-AAT glycofoms were analyzed by isoelectric focusing on agarose gels followed by immunofixation using the Hydragel

Figure 1. Isoelectric focusing patterns of AAT glycoforms. (A) Isoelectric-focusing patterns of AAT glycoforms for ZZ-AATD patient plasma (closed arrow)) compared to plasma of a MM-individual (open arrow) using the Hydragel AAT isofocusing kit (linear pH gradient of 4.2−4.9). The positive (+) anode and negative (−) cathode gel orientation is demonstrated. The Z-AAT glycoforms are located near the cathode (−) and are significantly fainter because of the low plasma AAT level. (B−D) Purified AAT (6 μg) was labeled with 0.4 mM CyDyes and added to IPG Strips (nonlinear gradient pH 3−5.6). (B) Cy3 labeled M-AAT; (C) Cy5 labeled Z-AAT; (D) Cy3 labeled MAAT overlaid with Cy5 labeled Z-AAT demonstrating cathodal shift of Z-AAT to the right of the gel. Results presented are representative gels from 5 separate experiments.

Figure 2. AAT HILIC chromatograms. (A) Typical HILIC chromatograms of undigested AAT from a healthy PiMM individual and PiZZ AATD individual. The HILIC chromatograms were separated into 16−18 peaks. The chromatograms shown in (A) are representative of a single glycoform from an individual 2-D separated spot from both an M-AAT sample and a Z-AAT sample and is representative of the glycan profile seen in M-AAT and Z-AAT. The glycans which are represented on each peak are identified in Figure 3. (B) Typical HILIC chromatograms of ABS digested AAT from a healthy PiMM individual and PiZZ AATD patient. The HILIC chromatograms were separated into 11 peaks. The glycans that are represented on each peak are identified in Figure 4 and the percentage areas of each peak from each individual spot of all patients and controls are detailed in Supporting Information Table S3.

AAT isofocusing kit (Sebia). This method separates various glycoforms of AAT on the basis of their isoelectric point in a pH gradient of between 4.2 and 4.9 and is routinely used for phenotyping of clinical samples. The different pattern of AAT glycoforms in plasma from MM and ZZ-AATD individuals is presented in Figure 1A. Consistently, a cathodal shift of three ZZ-AAT bands was observed compared to 6 M-AAT glycoforms. However, to fully understand the complexity of the differences between M- and Z-AAT and to observe the full spectrum of Z-AAT glycoforms, ensuing experiments employed 2D-DIGE over a pH range of 3−5.6. Cye-Dye labeling was carried out on purified AAT from all 10 male plasma samples: 5 AATD (Z-AAT) and 5 healthy controls (M-AAT). Between 8 and 9 glycoforms were identified on M-AAT (Figure 1B) and Z-AAT gels (Figure 1C). The Z-AAT demonstrated a 0.05− 0.13 pI cathodal shift of all glycoforms (Figure 1D). This shift is in keeping with previously published literature which suggests that the amino acid substitution of glutamate for lysine in ZAAT decreases the isoelectric point by 0.1.11 Conversely,

another study demonstrated that sialic acid residues on AAT can cause a cathodal shift ranging from 0.13 to 0.31 in pI.35,36 Therefore, ensuing experiments performed glycoanalysis of Mand Z-AAT cut from Coomassie blue-stained 2D SDS-PAGE gels separated on nonlinear pH gradients of 3−5.6. In all experiments the purity of M-AAT and Z-AAT was confirmed by mass spectrometry analysis. LC−MS/MS confirmed that spots on 2D SDS-PAGE gels were AAT with an average coverage of 64% in PiMM and 66% in PiZZ individuals using the MASCOT algorithm and an average coverage of 69% in PiMM and 61% in PiZZ individuals using the SEQUEST algorithm (Supporting Information Table S1). 599



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Figure 3. Summary of N-glycans identified in AAT. Monosaccharide residue symbols and bond angles of the pictured glycans are shown. The glycans presented represent the glycans for each of the 18 peaks shown on the chromatograms in Figure 2A. Structures highlighted in orange are core fucosylated; those in yellow are outer arm fucosylated, and those in green contain both core and outer arm fucose groups. Nomenclature used is according to Royle et al.31 and Harvey et al..53 All N-glycans have two core GlcNAcs; F at the start of the abbreviation indicates a core fucose α1,6linked to the inner GlcNAc; Mx, number (x) of mannose on core GlcNAcs; number of antenna (GlcNAc) on trimannosyl core; A2, biantennary with both GlcNAcs as β1,2-linked; A3, triantennary with a GlcNAc linked β1,2 to both mannose and the third GlcNAc linked β1,4 to the α1,3-linked mannose; A4, GlcNAcs linked as A3 with additional GlcNAc β1,6 linked to α1,6 mannose; A3′, isomer with the third GlcNAc linked β1−6 to the α1−6 linked mannose; B, bisecting GlcNAc linked β1,4 to β1,3 mannose; Gx, number (x) of β1,4-linked galactose on antenna; Sx, number (x) of sialic acids linked to galactose; the numbers 3 or 6 or in parentheses after S indicate whether the sialic acid is in an α2,3 or α2,6 linkage. If there is no linkage number, the exact link is unknown. 600



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Figure 4. Summary of N-glycans identified on ABS digested AAT. Monosaccharide residue symbols and bond angles of the pictured glycans are shown. The glycans presented represent the glycans for each of the 11 peaks shown on the chromatograms in Figure 2B. Structures highlighted in orange are core fucosylated; those in yellow are outer arm fucosylated, and those in green contain both core and outer arm fucose groups. Nomenclature used is according to Royle et al.31 and Harvey et al.53 and is the same as used in Figure 3. Peak areas from ABS digested chromatograms for all 37 M-AAT glycoforms and all 32 Z-AAT glycoforms are presented in Supporting Information Table S3.

No Significant Difference in the Branching of Glycans Was Found on M-AAT and Z-AAT

mono-, bi-, tri-, and tetra-sialylated, some containing core fucose (α1−6 linked) and/or outer arm fucose (α1−3 linked, forming Lewis x (Lex) epitopes, or together with sialic acids sialyl Lewis x (sLex) epitopes). The total levels of bi-, tri-, tetraantennary, core and outer arm fucosylated glycans (coming from digested Lex and sialyl Lewis x (sLex) epitopes) were quantified by digesting the glycan pool of each individual glycoform from all controls and patients with A. ureafaciens sialidase (ABS) (Supporting Information Table S3). There was no statistically significant difference between MAAT and Z-AAT when the percentages of biantennary glycans were compared, 77.97% and 78.43%, respectively (Figure 5A; p = 0.92). Similarly, no difference was found between the proportion of triantennary glycans, 19.54% vs 19.58% (Figure 5B; p = 0.77) or tetra-antennary glycans, 2.49% vs 1.98% (Figure 5C; p = 0.16). Collectively, these results indicate similar

The N-glycans of purified Z-AAT and M-AAT from 10 plasma samples were analyzed by UPLC in combination with exoglycosidase digestions and structural assignments as described by Royle et al.31 and the software tool GlycoBase (http://glycobase.nibrt.ie) (Supporting Information Table S2). The technical reproducibility of the glycan analysis was acceptable. The average deviation of peaks was below 10%. Peaks which were over 10% reproducibility were very small peaks at the beginning and at the end of profiles and of very low intensity (in 10 replicates). Typical HILIC chromatograms of undigested AAT from a healthy PiMM individual and a PiZZ AATD patient are shown in Figure 2 and the list of identified glycans is presented in Figures 3 and 4. Present were complex glycans which were bi-, tri-, and tetra-antennary; neutral; 601



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Figure 6. Percentages of fucosylated glycans on M-AAT and Z-AAT. (A) A significant difference in core fucosylation exists between M-AAT and Z-AAT. The percentage of glycans containing a core (α1,6 linked) fucose in all spots from M-AAT compared to all spots in Z-AAT samples. Mean of each group is indicated by the horizontal line and statistical significance calculated by Mann−Whitney test (p < 0.0001). (B) A significant difference exists in outer arm fucosylation between M-AAT and Z-AAT. The percentage of glycans containing an outer arm (α1,3 linked) fucose in all spots from M-AAT compared to all spots in Z-AAT samples. Mean of each group is indicated by the horizontal line and statistical significance was calculated by Mann− Whitney test (p = 0.04).

Figure 5. Percentages of branched glycans on M-AAT and Z-AAT. Percentage of (A) biantennary, (B) triantennary, and (C) tetraantennary glycans on M-AAT and Z-AAT. No significant difference (NS) was detected in the percentage of glycans that are branched in biantennary, triantennary, or tetra-antennary fashion on M-AAT and Z-AAT. Mean of each group is indicated by the horizontal line and statistical significance was calculated by Mann−Whitney test.

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DISCUSSION In this study, the N-glycans of AAT were comprehensively analyzed from 5 ZZ-AATD individuals and 5 healthy MMcontrols and this is the first complete glycoanalysis performed on Z-AAT. Clinical diagnosis of severe AATD is based on low plasma AAT levels and the presence of the PiZZ phenotype, as demonstrated by a cathodal shift of AAT on diagnostic IEF gels. In this study, we have confirmed the cathodal shift of ZAAT by 0.05 to 0.13 in pI. However, as the results have shown no significant difference in the glycan branching on Z-AAT compared to M-AAT, the cathodal shift of Z-AAT is most likely due to the amino acid substitution. Importantly, as Z-AAT expresses branched glycans similar to M-AAT, this may convey comparable anti-inflammatory effects to the Z-AAT protein. In this regard, we have previously shown that glycosylated AAT rather than deglycosylated AAT has a greater ability to modulate IL-8 activity.5 Thus, if Z-AAT has no significant change in charge due to branching, then it may possibly retain substantial immune modulatory properties. This preserved immune modulatory role may prove vital in encouraging studies investigating novel techniques to enhance secretion of Z-AAT from hepatocytes in AATD individuals, including the use of 6-mer peptides37,38 or chemical chaperones,39 despite what is known about Z-AAT having a reduced antineutrophil elastase capacity. Interestingly, in this study, we have identified a significant difference in the fucosylation pattern between M-AAT and ZAAT. There is increased total fucosylation in Z-AAT including

levels of bi-, tri-, and tetra-antennary glycan structures which may equally affect potential immuno-modulatory functions of M-AAT and Z-AAT. Alpha-1 Antitrypsin from PiZZ Deficient Individuals Contains More Core and Outer Arm Fucosylated Glycans

The most significant and notable difference in the glycosylation pattern between M-AAT and Z-AAT was the increased fucosylation of Z-AAT. Results revealed that core fucosylation was significantly greater in Z-AAT protein (range 3.5%10.02%), with an average of 6.65% (95% CI 6.05−7.24%) of all glycans containing a core fucose (α1,6) in Z-AAT compared to an average of 5.07% (95% CI 4.7−5.3%) in M-AAT (range 3.6−8%) (p < 0.0001, Figure 6A). Also, a significantly higher proportion of outer arm fucosylated (α1,3) glycans was detected in Z-AAT: on average 12.5% of glycans (95% CI 10.57−14.4%) contained an outer arm fucose (range 5.8− 32.8%) compared to an average of 9.5% (95% CI 8.08−10.9%) of M-AAT glycans (range 2.24−18.4%) (p = 0.04, Figure 6B). This increase in both core and outer arm fucosylation differentiates Z-AAT from M-AAT and may alter the immuno-regulatory capacity of Z-AAT. 602



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disease.4,5,50 This may have implications for initiating treatment earlier for seemingly healthy PiZZ individuals in order to reduce the reported subclinical inflammation.

both core fucosylation and outer arm fucosylation. Both of these increased fucosylation states raise interesting questions as to their effect on Z-AAT. Core fucosylation of circulating acute phase proteins has been shown previously to be increased in inflammatory conditions such as Crohn’s disease40 and malignant diseases such as lung cancer and pancreatic cancer when compared to healthy controls.41−43 Changes in core fucosylation have been shown to occur in a stepwise fashion in the progression from cirrhotic liver disease to hepatocellular carcinoma.44 Generally, with increasing age there is a decrease in core fucosylated glycans;45,46 however, in this study we have shown that there is an increase in core fucosylation in the PiZZ group despite the fact that the mean age was older than the control group. Moreover, in a study by Pucic et al. (2011) examining the changes in glycosylation of IgG with age, no change in core fucosylation with increasing age was observed.47 Furthermore, Knezevic et al. (2010) demonstrated that men aged 30−39 had no significant difference in core or outer arm fucosylation compared to men aged 40−49,48 thus again supporting the conclusion that the differences in fucosylation between Z-AAT and M-AAT is not as a result of age as our mean ages were 46 and 32 years, respectively. The increase in outer arm fucosylated glycans, which includes Lex type fucosylation, is of interest in Z-AAT. It has previously been shown that this type of fucosylation is increased in different acute phase proteins (including AAT) at times of acute inflammation and in chronic inflammatory conditions including rheumatoid arthritis, diabetes mellitus and chronic pancreatitis.41,42 Similarly, it has been shown how sLex type fucosylation may increase in circulating plasma proteins and subsequently act as a biomarker of lung cancer49 and other malignancies such as ovarian, breast and prostate.41 This raises the question as to why there is increased outer arm fucosylation in Z-AAT. One explanation could be the presence of continual inflammatory processes in AATD as previously described.4,5,50 Second, it may be due to the processing of mutant Z-AAT. Because of the conformational change secondary to the amino acid substitution, Z-AAT is processed in the endoplasmic reticulum and Golgi apparatus differently to M-AAT51 and this may in part explain increased levels of sLex type fucosylation. When considering the impact of increased sLex glycans, the latter has previously been shown to modulate attachment of leukocytes to E-selectins on endothelial membrane surfaces. Previous work in this field has shown that plasma serpin C1 inhibitor (C1INH) inhibits leukocyte adhesion through the interference of sLex tetrasaccharide with E and P selectins. This effect is lost when C1INH is deglycosylated, indicating that the primary mechanism through which it inhibits leukocyteendothelial adhesion is the presence of sLex and is independent of its antiprotease activity.52 This could have implications for plasma M-AAT as the presence of sLex may affect leukocyte adhesion and as Z-AAT is more fucosylated, the mutated Zprotein may exhibit an enhanced antiadhesive efficiency over M-AAT. In conclusion, this is the first comprehensive glycoanalysis of Z-AAT and comparison with M-AAT. This study demonstrates the significant increase in outer arm and core fucosylation of ZAAT, both of which may have implications for its function as an immune modulatory protein and its effect upon leukocyte mediated inflammation in AATD. Finally, our results support the hypothesis that AATD is an inflammatory disorder with chronic low grade inflammation present even in apparently asymptomatic individuals or individuals with early stage

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ASSOCIATED CONTENT

* Supporting Information S

Detailed methods and results of LC−MS/MS anlysis; purity of AAT before and after the enrichment procedure; summary of N-glycans identified in AAT; summary of peak areas following ABS digestion of each individual glycoform from all M-AAT and Z-AAT 2D -PAGE gels. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Emer P. Reeves, Ph.D., Tel.: +353-1-8093877. Fax: +353 1 8093808. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.G.M. and E.P.R. acknowledge funding from the US Alpha One Foundation and the Medical Research Charities Group/ Health Research Board Ireland and the Program for Research in Third Level Institutes (PRTLI) administered by the Higher Education Authority. P.M., M.C., N.G.M. and E.P.R. acknowledge funding from the 3U Biomedical Research (DCU-NUI Maynooth-RCSI). R.S. acknowledges funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 260600 (“GlycoHIT”). We thank Kitty O’Connor and Grace Mullins from the Alpha One Foundation Ireland for collection of patient samples.

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ABBREVIATIONS

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REFERENCES

2AB, 2-aminobenzamide; 2D-PAGE, 2-dimensional polyacrylamide gel electrophoresis; 2D-DIGE, 2D-fluorescence difference gel electrophoresis; AAT, alpha-1 antitrypsin; AATD, alpha-1 antitrypsin deficiency; ABS, Arthrobacter ureafaciens sialidase; AMF, almond meal α-fucosidase; BKF, bovine kidney αfucosidase; BTG, bovine testes β-galactosidase; C1INH, plasma C1 inhibitor; DTT, dithiothreitol; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; GU, glucose unit; GUH, β-N-acetylglucosaminidase cloned from Streptococcus pneumoniae, expressed in E. coli; HILIC, hydrophilic interaction liquid chromatography; IEF, isoelectric focusing; IPG, immobilized pH gradient; LC, liquid chromatography; MS, mass spectrometry; NAN1, Streptococcus pneumoniae sialidase; NE, neutrophil elastase; NL, nonlinear; RCL, reactive center loop; RPM, revolutions per minute; TFA, trifluoroacetic acid; UPLC, ultra performance liquid chromatography; CI, confidence interval

(1) Donato, L. J.; Jenkins, S. M.; Smith, C.; Katzmann, J. A.; Snyder, M. R. Reference and interpretive ranges for alpha(1)-antitrypsin quantitation by phenotype in adult and pediatric populations. Am. J. Clin. Pathol. 2012, 138 (3), 398−405. (2) Eriksson, S.; Alm, R.; Astedt, B. Organ cultures of human fetal hepatocytes in the study of extra-and intracellular alpha1-antitrypsin. Biochim. Biophys. Acta 1978, 542 (3), 496−505.

603



Article

antitrypsin under anti-inflammatory conditions. Cell Transplant. 2013, 22 (11), 2119−33. (23) de Serres, F. J. Worldwide racial and ethnic distribution of alpha1-antitrypsin deficiency: summary of an analysis of published genetic epidemiologic surveys. Chest 2002, 122 (5), 1818−29. (24) Foreman, R. C. Alpha 1-antitrypsin deficiencya defect in secretion. Biosci. Rep. 1987, 7 (4), 307−11. (25) Lomas, D. A.; Evans, D. L.; Finch, J. T.; Carrell, R. W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 1992, 357 (6379), 605−7. (26) Lomas, D. A.; Evans, D. L.; Stone, S. R.; Chang, W. S.; Carrell, R. W. Effect of the Z mutation on the physical and inhibitory properties of alpha 1-antitrypsin. Biochemistry 1993, 32 (2), 500−8. (27) Jeppsson, J. O.; Larsson, C.; Eriksson, S. Characterization of alpha1-antitrypsin in the inclusion bodies from the liver in alpha 1antitrypsin deficiency. N. Engl. J. Med. 1975, 293 (12), 576−9. (28) Cowden, D. I.; Fisher, G. E.; Weeks, R. L. A pilot study comparing the purity, functionality and isoform composition of alpha1-proteinase inhibitor (human) products. Curr. Med. Res. Opin. 2005, 21 (6), 877−83. (29) Lindhout, T.; Iqbal, U.; Willis, L. M.; Reid, A. N.; Li, J.; Liu, X.; et al. Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (18), 7397−402. (30) Zerimech, F.; Hennache, G.; Bellon, F.; Barouh, G.; Lafitte, J. J.; Porchet, N.; et al. Evaluation of a new Sebia isolectrofocusing kit for a1-antitrypsin phenotyping with the Hydrasys System. Clin. Chem. Lab. Med. 2008, 46 (2), 260−3. (31) Royle, L.; Campbell, M. P.; Radcliffe, C. M.; White, D. M.; Harvey, D. J.; Abrahams, J. L.; et al. HPLC-based analysis of serum Nglycans on a 96-well plate platform with dedicated database software. Anal. Biochem. 2008, 376 (1), 1−12. (32) Kuster, B.; Wheeler, S. F.; Hunter, A. P.; Dwek, R. A.; Harvey, D. J. Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase highperformance liquid chromatography. Anal. Biochem. 1997, 250 (1), 82−101. (33) Bigge, J. C.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal. Biochem. 1995, 230 (2), 229−38. (34) Royle, L.; Radcliffe, C. M.; Dwek, R. A.; Rudd, P. M. Detailed structural analysis of N-glycans released from glycoproteins in SDSPAGE gel bands using HPLC combined with exoglycosidase array digestions. Methods Mol. Biol. 2006, 347, 125−43. (35) Barrabes, S.; Sarrats, A.; Fort, E.; De Llorens, R.; Rudd, P. M.; Peracaula, R. Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresis. Electrophoresis 2010, 31 (17), 2903−12. (36) Wilson, N. L.; Schulz, B. L.; Karlsson, N. G.; Packer, N. H. Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins. J.Proteome Res. 2002, 1 (6), 521−9. (37) Mallya, M.; Phillips, R. L.; Saldanha, S. A.; Gooptu, B.; Brown, S. C.; Termine, D. J.; et al. Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates. J. Med. Chem. 2007, 50 (22), 5357−63. (38) Parfrey, H.; Dafforn, T. R.; Belorgey, D.; Lomas, D. A.; Mahadeva, R. Inhibiting polymerization: new therapeutic strategies for Z alpha1-antitrypsin-related emphysema. Am. J. Respir. Cell Mol. Biol. 2004, 31 (2), 133−9. (39) Burrows, J. A.; Willis, L. K.; Perlmutter, D. H. Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (4), 1796−801. (40) Goodarzi, M. T.; Turner, G. A. Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by

(3) Cichy, J.; Potempa, J.; Travis, J. Biosynthesis of alpha1-proteinase inhibitor by human lung-derived epithelial cells. J. Biol. Chem. 1997, 272 (13), 8250−5. (4) Carroll, T. P.; Greene, C. M.; O’Connor, C. A.; Nolan, A. M.; O’Neill, S. J.; McElvaney, N. G. Evidence for unfolded protein response activation in monocytes from individuals with alpha-1 antitrypsin deficiency. J. Immunol. 2010, 184 (8), 4538−46. (5) Bergin, D. A.; Reeves, E. P.; Meleady, P.; Henry, M.; McElvaney, O. J.; Carroll, T. P.; et al. Alpha-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL8. J. Clin. Invest. 2010, 120 (12), 4236−50. (6) du Bois, R. M.; Bernaudin, J. F.; Paakko, P.; Hubbard, R.; Takahashi, H.; Ferrans, V.; et al. Human neutrophils express the alpha 1-antitrypsin gene and produce alpha 1-antitrypsin. Blood 1991, 77 (12), 2724−30. (7) Molmenti, E. P.; Perlmutter, D. H.; Rubin, D. C. Cell-specific expression of alpha 1-antitrypsin in human intestinal epithelium. J. Clin. Invest. 1993, 92 (4), 2022−34. (8) Chen, X. L.; Zhou, L.; Yang, J.; Shen, F. K.; Zhao, S. P.; Wang, Y. L. Hepatocellular carcinoma-associated protein markers investigated by MALDI-TOF MS. Mol. Med. Rep. 2010, 3 (4), 589−96. (9) Kolarich, D.; Turecek, P. L.; Weber, A.; Mitterer, A.; Graninger, M.; Matthiessen, P.; et al. Biochemical, molecular characterization, and glycoproteomic analyses of alpha(1)-proteinase inhibitor products used for replacement therapy. Transfusion 2006, 46 (11), 1959−77. (10) Kolarich, D.; Weber, A.; Turecek, P. L.; Schwarz, H. P.; Altmann, F. Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms. Proteomics 2006, 6 (11), 3369−80. (11) Mills, K.; Mills, P. B.; Clayton, P. T.; Johnson, A. W.; Whitehouse, D. B.; Winchester, B. G. Identification of alpha(1)antitrypsin variants in plasma with the use of proteomic technology. Clin. Chem. 2001, 47 (11), 2012−22. (12) Duranton, J.; Bieth, J. G. Inhibition of proteinase 3 by [alpha]1antitrypsin in vitro predicts very fast inhibition in vivo. Am. J. Respir. Cell Mol. Biol. 2003, 29 (1), 57−61. (13) Elliott, P. R.; Lomas, D. A.; Carrell, R. W.; Abrahams, J. P. Inhibitory conformation of the reactive loop of alpha 1-antitrypsin. Nat. Struct. Biol. 1996, 3 (8), 676−81. (14) Elliott, P. R.; Pei, X. Y.; Dafforn, T. R.; Lomas, D. A. Topography of a 2.0 Å structure of alpha1-antitrypsin reveals targets for rational drug design to prevent conformational disease. Protein Sci. 2000, 9 (7), 1274−81. (15) Desgranges, S.; Le Prieult, F.; Daly, A.; Lydon, J.; Brennan, M.; Rai, D. K.; et al. In vitro activities of synthetic host defense propeptides processed by neutrophil elastase against cystic fibrosis pathogens. Antimicrob. Agents Chemother. 2011, 55 (5), 2487−9. (16) Huber, R.; Carrell, R. W. Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry 1989, 28 (23), 8951−66. (17) Kushner, I. The phenomenon of the acute phase response. Ann. N.Y. Acad. Sci. 1982, 389, 39−48. (18) Perlmutter, D. H. Alpha-1-antitrypsin deficiency: diagnosis and treatment. Clin. Liver Dis. 2004, 8 (4), 839−59. (19) Petrache, I.; Fijalkowska, I.; Zhen, L.; Medler, T. R.; Brown, E.; Cruz, P.; et al. A novel antiapoptotic role for alpha1-antitrypsin in the prevention of pulmonary emphysema. Am. J. Respir. Crit. Care Med. 2006, 173 (11), 1222−8. (20) Al-Omari, M.; Korenbaum, E.; Ballmaier, M.; Lehmann, U.; Jonigk, D.; Manstein, D. J.; et al. Acute-phase protein alpha1antitrypsin inhibits neutrophil calpain I and induces random migration. Mol. Med. 2011, 17 (9−10), 865−74. (21) Petrache, I.; Fijalkowska, I.; Medler, T. R.; Skirball, J.; Cruz, P.; Zhen, L.; et al. Alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis. American J. Pathol. 2006, 169 (4), 1155−66. (22) Bellacen, K.; Kalay, N.; Ozeri, E.; Shahaf, G.; Lewis, E. C. Revascularization of pancreatic islet allografts is enhanced by alpha-1604



Article

PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease. Glycoconjugate J. 1998, 15 (5), 469−75. (41) Peracaula, R.; Sarrats, A.; Rudd, P. M. Liver proteins as sensor of human malignancies and inflammation. Proteomics: Clin. Appl. 2010, 4 (4), 426−31. (42) Sarrats, A.; Saldova, R.; Pla, E.; Fort, E.; Harvey, D. J.; Struwe, W. B.; et al. Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis. Proteomics: Clin. Appl. 2010, 4 (4), 432−48. (43) Ueda, K.; Katagiri, T.; Shimada, T.; Irie, S.; Sato, T. A.; Nakamura, Y.; et al. Comparative profiling of serum glycoproteome by sequential purification of glycoproteins and 2-nitrobenzensulfenyl (NBS) stable isotope labeling: a new approach for the novel biomarker discovery for cancer. J. Proteome Res. 2007, 6 (9), 3475−83. (44) Comunale, M. A.; Rodemich-Betesh, L.; Hafner, J.; Wang, M.; Norton, P.; Di Bisceglie, A. M.; et al. Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and cancer patients: implications for a biomarker of hepatocellular carcinoma. PLoS One 2010, 5 (8), e12419. (45) Knezevic, A.; Polasek, O.; Gornik, O.; Rudan, I.; Campbell, H.; Hayward, C.; et al. Variability, heritability and environmental determinants of human plasma N-glycome. J. Proteome Res. 2009, 8 (2), 694−701. (46) Yamada, E.; Tsukamoto, Y.; Sasaki, R.; Yagyu, K.; Takahashi, N. Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum. Glycoconjugate J. 1997, 14 (3), 401−5. (47) Pucic, M.; Knezevic, A.; Vidic, J.; Adamczyk, B.; Novokmet, M.; Polasek, O.; et al. High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations. Mol. Cell. Proteomics 2011, 10 (10), No. M111.010090. (48) Knezevic, A.; Gornik, O.; Polasek, O.; Pucic, M.; Redzic, I.; Novokmet, M.; et al. Effects of aging, body mass index, plasma lipid profiles, and smoking on human plasma N-glycans. Glycobiology 2010, 20 (8), 959−69. (49) Arnold, J. N.; Saldova, R.; Galligan, M. C.; Murphy, T. B.; Mimura-Kimura, Y.; Telford, J. E.; et al. Novel glycan biomarkers for the detection of lung cancer. J. Proteome Res. 2011, 10 (4), 1755−64. (50) Rouhani, F.; Paone, G.; Smith, N. K.; Krein, P.; Barnes, P.; Brantly, M. L. Lung neutrophil burden correlates with increased proinflammatory cytokines and decreased lung function in individuals with alpha(1)-antitrypsin deficiency. Chest 2000, 117 (5 Suppl. 1), 250S−1S. (51) Cabral, C. M.; Liu, Y.; Moremen, K. W.; Sifers, R. N. Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs. Mol. Biol. Cell 2002, 13 (8), 2639−50. (52) Cai, S.; Dole, V. S.; Bergmeier, W.; Scafidi, J.; Feng, H.; Wagner, D. D.; et al. A direct role for C1 inhibitor in regulation of leukocyte adhesion. J. Immunol. 2005, 174 (10), 6462−6. (53) Harvey, D. J.; Merry, A. H.; Royle, L.; Campbell, M. P.; Dwek, R. A.; Rudd, P. M. Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics 2009, 9 (15), 3796−801.

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Increased Outer Arm and Core Fucose Residues on the N-Glycans of

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