Research Communication

Physicochemical Studies on Glycation-induced StructuralChanges in Human IgG

Saman Ahmad1, Moinuddin1, Rizwan Hasan Khan2,3 and Asif Ali11Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, UP, India2Department of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK3Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, India

Summary

Glycation of biomolecules leads to the formation of advancedglycation end products (AGEs). Glycation of immunoglobulin G(IgG) has been implicated in autoimmune diseases such as rheu-matoid arthritis (RA). In this study, human IgG was glycatedwith physiological concentration of glucose. The changesinduced in IgG were analyzed by UV, fluorescence, circulardichroism, and Fourier transform infrared (FTIR) spectros-copy; thermal denaturation studies, native, and Sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis. The keto-amine moieties and carbonyl content were also quantitated inglycated IgG. We report structural perturbations, increasedcarbonyl content, and ketoamine moieties in the glycated IgG.This may interfere with the normal function of IgG and maycontribute to initiation of arthritic complications. AGEs dam-aged IgG may be used as a biomarker for early detection ofRA and the associated secondary complications. � 2012 IUBMB

IUBMB Life, 64(2): 151–156, 2012

Keywords glycation; IgG; spectroscopy; AGEs; glucose.

INTRODUCTION

Glycation is the nonenzymatic addition of reducing sugars to

biological macromolecules. Carbonyl groups of reducing sugars

readily react with the free amino groups of proteins to form

Schiff base that undergoes rearrangement forming a more stable

early glycation product known as Amadori product (ketoamine)

(1) which finally forms advanced glycation end products

(AGEs) (2). AGEs alter the three-dimensional integrity of vari-

ous plasma proteins which could induce functional abnormal-

ities and thereby lead to pathogenesis of several diseases (3).

AGEs formation is accelerated in the pathophysiological condi-

tions of oxidative stress and/or hyperglycemia. Physiological

AGE formation from the stable ketoamine glycated protein has

been correlated with the oxidative and inflammatory processes

and not with glucose levels (4). Only few studies have reported

the role of oxidative stress and AGEs in the inflammatory activ-

ity in rheumatoid arthritis (RA) (5–8), whereas the majority of

work has focused on diabetes and hyperglycemic conditions.

Nonenzymatic glycosylation of a variety of plasma proteins

has been shown to occur in normoglycemic individuals in vivo.

The differences in glycation among individual plasma proteins are

extreme. The dominant factor in protein glycation is the half-life

of individual proteins; greater the half-life, greater the glycation

(9, 10). IgG and albumin having half-lives of 24 days and 15–19

days, respectively, exhibit maximum in vivo glycation. However,

at high glucose concentration, the extent of glycation is mainly

determined by intrinsic glycability of protein. For instance, when

plasma proteins were glycated in vitro by 0.5 mol/L glucose, IgG

showed greater extent of glycation followed in descending order

by complement C3 (half-life: 2–5 days), albumin (15–19 days),

transferrin (7 days), haptoglobin (2 days), and a-1-antitrypsin (4

days). As complement C3 has high intrinsic glycability, so it

showed high degree of glycation, despite a short half-life (9).

IgG constitutes about 75% of the total immunoglobulin pool

in serum. In addition to four N-terminal amino acids, human

IgG1 has �80 lysine residues, making IgG a good target for

glycation (11). Glycation of IgG is of special interest due to its

influence on the functionality of immunoglobulins and overall

immunocompetence, especially with regard to their ability to

bind antigens and induce the complement cascade. Glycation of

immunoglobulins has been shown to cause major structural per-

turbations resulting in their functional disability (12–15). While

hemoglobin, albumin, and collagen are common targets of AGE

modification in diabetes (16), glycated IgG (AGE-IgG) is asso-

ciated with inflammation in RA (17). Newkirk et al. (18)

Address correspondence to: Asif Ali, Department of Biochemistry,

Faculty of Medicine, J.N. Medical College, Aligarh Muslim University,

Aligarh 202002, India. Tel: 191-941-227-3580. Fax: 191-571-272-

0030. E-mail: asifali_amu@rediffmail.com

Received 11 May 2011; accepted 4 September 2011

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.582

IUBMB Life, 64(2): 151–156, February 2012

showed that AGE-IgG is a target for circulating autoantibodies

in RA patients. Ligier et al. (19) have reported AGE-IgG and

elevated titers of IgM and IgA antibodies against AGE-IgG in

RA patients with severe systemic manifestations.

In this study, in vitro glycation of IgG was done using physi-

ological concentration of glucose (5 mM).

EXPERIMENTAL

Glycation

Human IgG (Sigma, St. Louis, MO) at a concentration of

0.825 lM in Phosphate buffered saline (PBS) (10-mM sodium

phosphate buffer, 150 mM NaCl, and pH 7.4) was incubated

with 5 mM glucose (SRL, India) under sterile conditions at 37

8C for 5–25 days. After incubation, the solutions were exten-

sively dialyzed against PBS to remove excess glucose.

Absorbance Spectroscopy

The absorption profiles of native and glycated samples were

recorded on Shimadzu UV 1700 spectrophotometer in the wave-

length range of 200–400 nm.

Electrophoresis

Native and glycated IgG were analyzed by native and So-

dium dodecyl sulphate-Polyacrylamide gel electrophoresis

(SDS-PAGE) (in nonreducing conditions) using 10% polyacryl-

amide gel as described previously (20), and the protein bands

were visualized by silver staining.

Fluorescence Studies

Fluorescence spectra were recorded on Hitachi F-200 spec-

trofluorimeter (Japan) at 25 6 0.1 8C. Fluroscence of trypto-

phan residues in native and glycated samples was monitored

with excitation at 295 nm and measuring emission in 300–400

nm range. Loss in the fluorescence intensity (FI) was calculated

from the following equation:

% loss of FI ¼ ½ðFInative sample �FIglycated sampleÞ=FInative sample�3100

Possible presence of fluorogenic AGEs in the glycated sam-

ple was verified with AGE-specific fluorescence emission at

440 nm after excitation at 370 nm (21). Increase in FI was

calculated from the following equation:

% increase of FI ¼ ½ðFIglycated sample �FIInative sampleÞ=FIglycated sample�3100

Circular Dichroism Measurements

Far-UV Circular dichroism (CD) measurements of native and

glycated IgG samples (2.5 lM) were recorded on J-815 Jasco

spectropolarimeter in the wavelength range of 200–250 nm

(22). All scans were recorded at wavelength intervals of 1 nm.

FTIR Spectroscopy

For FTIR analysis, native and glycated IgG samples were

first lyophilized and prepared as KBr pellets and then subjected

to spectral recording on a Shimadzu FTIR spectrophotometer

(8201-PC).

Thermal Denaturation

Thermal denaturation of native and glycated IgG was moni-

tored at 280 nm in the temperature range of 30 to 90 8C on Shi-

madzu UV-1700 spectrophotometer equipped with a thermo-

programmer and a controller unit.

Determination of Protein-bound Carbonyl Groups

Carbonyl contents of native and glycated IgG samples was

determined using 2,4-dinitrophenylhydrazine (23). The absorb-

ance was read at 360 nm and the carbonyl content determined

using extinction coefficient of 22,000 M21 cm21.

NBT Reduction Assay

The ketoamine moieties formed upon IgG glycation were

determined by nitroblue tetrazolium (NBT) reduction assay as

described previously (24). The absorbance of protein samples

was recorded at 525 nm. The content of ketoamine moieties

(nmol mL21) was determined using an extinction coefficient of

12640 M21 cm21.

Thiobarbituric Acid Assay

Hydroxymethylfurfural (HMF) in native and glycated IgG

samples was estimated by thiobarbituric acid assay as described

earlier (25).

Statistical Analysis

Data are presented as mean 6 Standard deviation (SD). Sta-

tistical significance of data was determined by Student’s t test,

and a p value of\0.05 was considered as significant.

RESULTS

Native IgG showed an absorbance peak at 278 nm which

increased on modification by glucose (Fig. 1). Compared to

native IgG, the hyperchromicities shown by glycated IgG incu-

bated for 5, 10, 15, and 20 days were 12.6, 26.6, 39.8, and

52.1%, respectively. However, after 25 days of incubation, gly-

cated sample showed 54.1% hyperchromicity indicating the sat-

uration limit of glycation.

Glycated IgG samples were further analyzed by native and

nonreducing SDS-PAGE. Sample incubated for 5 days did not

show any change in mobility and band intensity when compared

to the native protein. However, an increase in mobility toward

the anode (Fig. 2A) as well as decrease in band intensity and

appearance of multiple bands was observed with increasing

incubation time (Fig. 2B). Furthermore, 20 and 25 days incuba-

tion samples showed almost identical pattern on native poly-

152 AHMAD ET AL.

acrylamide gel electrophoresis (PAGE) which may be due to

generation of nearly equal net charge on the IgG molecule. The

appearance of multiple bands on SDS-PAGE is a clear indica-

tion of protein fragmentation on glycation. Not much difference

in the band pattern for 20 and 25 days incubation samples indi-

cates saturation limit for glycation. Therefore, further studies

were carried out with IgG glycated for 20 days (AGE-IgG).

Quenching of tryptophan fluorescence is measured as an

index of alteration in the tertiary structure of protein (26). In

our study, 53.1% loss in tryptophan fluorescence was seen at

295 nm on glycation (Fig. 3A).

The formation of AGEs was confirmed by fluorescence spec-

troscopy by exciting AGE-IgG at 370 nm. Under identical con-

ditions, native IgG does not give any fluorescence. Formation

of AGEs in AGE-IgG sample is characterized by emission max-

ima at 435 nm (Fig. 3B). An increase of 84.2% in FI was

observed in AGE-IgG when compared to the native form.

Figure 2. (A) Native polyacrylamide gel electrophoresis. Pro-

tein samples (5 lg per lane) were loaded onto 10% polyacryl-

amide gel. Lane 1: native IgG; lanes 2–6: IgG glycated with

glucose for 5, 10, 15, 20, and 25 days, respectively. (B) SDS-

polyacrylamide gel electrophoresis under nonreducing condi-

tions. Protein samples (5 lg per lane) were loaded on 10%

polyacrylamide gel. Lanes: (1) Protein molecular weight

markers; (2) Native IgG; (3–7) IgG glycated with glucose for 5,

10, 15, 20, and 25 days, respectively.

Figure 3. (A) Fluorescence emission spectra of native (—) and

AGE-IgG (---). Excitation wavelength was 295 nm. The spectra

are the average of three determinations. (B) Fluorescence emission

spectra of native (—) and AGE-IgG (---). Excitation wavelength

was 370 nm. The spectra are the average of three determinations.

Figure 1. Absorbance profile of native IgG (-&-) and IgG gly-

cated for 5 (-^-), 10 (-~-), 15(-^-), 20 (-n-), and 25 days

(-l-). The spectra are the average of three determinations.

153GLYCATION-INDUCED STRUCTURAL CHANGES IN HUMAN IgG

CD spectroscopy of native IgG showed characteristic spec-

trum of a primarily b-sheeted protein with negative minima at

217 nm and zero intensity at 206 nm. However, CD spectrum

of AGE-IgG showed increase in the ellipticity at 217 nm (Fig.

4), indicating a significant loss of b structure on glycation.

The FTIR spectra of native and AGE-IgG were analyzed for

the position of amide I and amide II bands. On glycation of

IgG, the peak position of amide I band got shifted from 1639.2

to 1627.8 cm21 and for amide II band, from 1550.6 to 1541.3

cm21 (Fig. 5). These changes demonstrated an alteration in the

IgG structure on glycation.

Thermal denaturation of native and AGE-IgG was investi-

gated between 30 and 90 8C. Change in absorbance was moni-

tored at 280 nm as a function of temperature, and percent dena-

turation was calculated. The melting temperature (Tm) of native

and AGE-IgG was found to be 70.5 and 74 8C respectively,

showing glycation-induced thermostability of AGE-IgG as com-

pared to its native analog (Fig. 6).

Oxidation of proteins typically results in an increase in protein

carbonyl contents, a known biomarker of oxidative stress. The

average carbonyl content (6SD) of five independent assays of

native and AGE-IgG were 8.3 6 1.3 nmol mg21 and 25.3 6 1.5

nmol mg21 protein, respectively (Table 1). This corresponds to

almost threefold increase in carbonyl level compared to native

IgG. For the detection of early glycation products, the ketoamine

moieties formed by glycation of IgG were measured colorimetri-

cally by NBT assay. Native IgG alone showed almost negligible

amount of ketoamine content, whereas glycated IgG had maxi-

mum ketoamine content at sixth day of incubation (Table 1). The

average ketoamine content (6SD) of five independent assays of

native and glycated IgG (at sixth day of incubation) were 1.66 6

0.5 nmol mL21 and 12.025 6 0.7 nmol mL21 protein, respec-

tively. Similarly, HMF that might have formed in the early gly-

cation of IgG was determined as thiobarbituric acid reactive sub-

stance after hydrolysis. The HMF content in glycated IgG was

also maximum at sixth day (8.125 6 0.6 nmol mL21 of protein),

whereas native IgG had almost negligible level of HMF (1.05 6

0.4 nmol mL21) (Table 1). It showed that early glycation was

maximum at sixth day and after that AGEs production would

start which leads to decrease in the ketoamine and HMF con-

tents. A p value of \0.001 indicates significant difference in the

carbonyl, ketoamine, and HMF contents of native and glycated

IgG samples. Characterization of native and glycated IgG has

been summarized in Table 1.

DISCUSSION

IgGs are major serum proteins and are rich in lysine residues

making them highly potential target for glycation. Several stud-

Figure 4. Far UV CD spectra of native (—) and AGE-IgG (---).

The spectra are the average of three determinations.

Figure 5. FTIR spectra of (A) native and (B) AGE-IgG. The

spectra are the average of three determinations.

154 AHMAD ET AL.

ies have reported the glycation of IgG and its influence on bio-

logical functions of IgG (27).

In this study, glucose modified human IgG exhibited exten-

sive damage on analysis by various physicochemical techniques.

The glycated samples showed hyperchromicity at 278 nm, com-

pared to native IgG, which may be attributed to exposure of

chromophoric aromatic amino acid residues due to the unfold-

ing and fragmentation of protein as a result of glycation (28).

Increased electrophoretic mobility in native PAGE on glycation

due to neutralization of positively charged residues in the pro-

tein revealed significant structural perturbation in IgG when

compared with native form. The SDS-PAGE of glycated sam-

ples showed noticeable time-dependent decrease in the intensity

of bands and appearance of two bands due to the glucose-

induced fragmentation of protein (29). The native and SDS-

PAGE pattern of AGE-IgG corroborated the spectral findings.

Loss of tryptophan fluorescence is ascribed to the destruction

of tryptophan and/or modification of tryptophan microenviron-

ment upon glycation (30). Fluorescence emission at 435 nm sig-

naled the formation of AGEs after excitation at 370 nm (31).

Native IgG showed a large negative dichroism at 217 nm in CD

spectroscopy which is a characteristic feature of b-sheet structure(32). Increase in ellipticitiy at 217 nm in AGE-IgG indicated a

substantial loss of b structure of IgG. Previous studies have

reported a loss of b-sheet fraction due to unfolding of IgG (33)

which confirms our findings. FTIR spectra clearly showed a shift

in amide I and amide II bands in the AGE-IgG when compared

with its native conformer, which is due to perturbation in the sec-

ondary structure of protein on glycation (34). Thermal denatura-

tion studies demonstrated that AGE-IgG was thermodynamically

more stable when compared with the native IgG. Formation of

cross-links and/or disulphide bridges could be the main reason

for the increased thermostability of AGE-IgG.

The maximal formation of ketoamine, assayed by NBT

reduction, was attained during the initial incubation itself, as

they are early nonenzymatic glycation adducts and are impor-

tant precursors of AGEs (35). The higher yield of HMF at sixth

day of incubation in glycated sample is in agreement with the

NBT assay result. Ketoamines are converted to protein carbonyl

compounds via a protein enediol generating superoxide radical

(36). Protein carbonyl content is most commonly used bio-

marker of protein oxidation and AGEs formation (23). The oxi-

dation of AGE-IgG was further evident by the significant

increase in its carbonyl content in comparison to native IgG.

The results reported here clearly demonstrate that in vitro

treatment of human IgG with glucose causes biophysical and

biochemical alterations resulting in the formation of AGEs. Fur-

ther studies are going on in our laboratory on AGE-IgG which

may play an important role in the induction of circulating auto-

antibodies in RA.

ACKNOWLEDGEMENTS

This study was supported by a research grant (62/3/2008-BMS)

from the Indian Council of Medical Research, New Delhi.

DST-FIST infrastructure facilities are also duly acknowledged.

Encouragement and help during the course of work by Dr.

Kiran and Dr. Saheem are duly acknowledged.

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Characterization of native and AGE-IgG under identical

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