Menu
Home
Log in / Register
 
Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Source

Glycation of Proteins

Naila Rabbani1 and Paul J. Thornalley1,2

  • 1 Warwick Systems Biology Centre, Coventry House, University of Warwick, Coventry, UK
  • 2 Warwick Medical School, Clinical Sciences Research Laboratories, University of Warwick, University Hospital, Coventry, UK

Overview of Protein Glycation

Protein glycation is a spontaneous post-translational modification (PTM) of proteins found in physiological systems and food products. It involves the nonenzymatic covalent attachment of a reducing sugar or sugar derivative to a protein [1]. It is a PTM that is often thermally and chemically labile, particularly at high pH and temperature when removed from the physiological setting. Analysis of protein glycation is compromised by the use of heating and high pH in preanalytic processing for mass spectrometric analysis. This is, perhaps, one of the most important points for experts in mass spectrometry new to protein glycation to consider and adapt experimental protocols accordingly for reliable analysis [2]. Initially, protein glycation was thought to be restricted to the modification of amino groups of lysine residue side chains and N-terminal amino acid residues by glucose. In more recent times, glycation of arginine residues by dicarbonyl metabolites has emerged as a major feature of protein glycation in physiological systems and linked to functional impairment [3]. There is also minor involvement of cysteine residues. This chapter mainly focuses on the detection and quantitation of the major early glycation adduct, ^e-(1-deoxy-D- fructos-1-yl)lysine of fructosyl-lysine (FL), and the major advanced glycation end product (AGE), methylglyoxal (MG)-derived hydroimidazolone MG-H1, and related compounds.

Protein glycation finds application in clinical metabolic monitoring and diagnosis. Glycation of albumin and hemoglobin by glucose produces glycated derivatives, glycated albumin, and glycated hemoglobin, used clinically in the assessment of glycemic control in diabetes over the 3-4 weeks and 6-8 weeks

Analysis of Protein Post-Translational Modifications by Mass Spectrometry, First Edition. Edited by John R. Griffiths and Richard D. Unwin.

© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

prior to sampling [4]. A defined range of glycated hemoglobin, 39-47 mmol/ mol Hb, intermediate between those found in healthy people and patients with diabetes is a diagnostic indicator of impaired glucose tolerance preceding diabetes and is used clinically in diagnostic screening for prediabetes [5]. Formation of AGEs is implicated in aging - including age-related macular degeneration and cataract, obesity, vascular complications of diabetes (nephropathy, retinopathy, and neuropathy), cirrhosis, cardiovascular disease, renal failure, and neurological disorders (Alzheimer's disease and Parkinson's disease) [6-17].

Glycation of proteins occurs by a complex series of sequential and parallel reactions called collectively the Maillard reaction. Many different adducts may be formed - some of which are fluorescent and colored “browning pigments.” In the physiological setting, one of the most important saccharides participating in glycation is glucose - forming lysine and N-terminal residue-derived fructosamine derivatives, and one of the most important saccharide derivatives is the reactive dicarbonyl metabolites MG, forming mainly arginine- derived hydroimidazolone derivatives [1].

Glycation adducts are classified into two groups: early-stage glycation adducts and AGEs. Glucose reacts with amino groups of lysine residue side chains and N-terminal amino acid residues to form sequentially a Schiff's base and then, via the Amadori rearrangement, NE-(1-deoxy-D-fructos-1-yl)lysine (FL) and Na-(1-deoxy-D-fructos-1-yl)amino acid residues - called collectively fructosamines (Figure 8.1). These are early-stage glycation adducts.

Examples of proteins susceptible to fructosamine formation are given in Table 8.1. Collectively these constitute the “fructosamine proteome” Schiff's base adducts are usually a minor component of glucose adducts in situ, ca. 10% of the level of FL residues in the steady state. They are also relatively rapidly reversed during sample isolation and processing, whereas fructosamines have much slower reversibility of formation; chemical relaxation times for reversal of Schiff's base and fructosamine formation are ca. 2.5 and 38 h at pH 7.4 and 37 °C, respectively [45]. The rate of fructosamine degradation at 37 °C increases markedly above pH 8 through increased reversal of the Amadori rearrangement and oxidative degradation to NE-carboxymethyl-lysine (CML) and related Na- carboxymethyl amino acids [45, 46]. Accordingly when adducts of early-stage glycation by glucose are detected and quantified, it is typically the fructosamine proteome that is characterized. Fructosamine modification of proteins is usually low, 5-10% modified protein, and may often have only moderate functional effects. This may be related to the respective low and moderate probability of location of N-terminal and lysine residues in the functional domains of proteins [47] and that the fructosamine residue retains the positive charge of the precursor lysyl side chain or N-terminal amino acid residue under physiological conditions. Gene knockout of fructosamine-3-phosphokinase (F3PK), an enzyme that repairs the fructosamine residues of cellular proteins, leads to accumulation of the fructosamine proteome without significant health impairment [48].

Major protein glycation processes in physiological systems. (a) Early glycation. Formation of the Schiff's base and fructosamine (Amadori product) of lysine residues

Figure 8.1 Major protein glycation processes in physiological systems. (a) Early glycation. Formation of the Schiff's base and fructosamine (Amadori product) of lysine residues.

(b) Oxidative degradation of fructosyl-lysine to W?-carboxymethyl-lysine. Similar processes occur on N-terminal amino acid residues. (c) Glycation of arginine residues by methylglyoxal with the formation of dihydroxyimidazolidine and hydroimidazolone MG-H1 residues. There are related structural isomers and similar adducts formed from glyoxal and 3-deoxyglucosone [1, 18-20].

In later-stage reactions of glycation, Schiff's base and fructosamine adducts degrade to form many stable end-stage adducts or AGEs [1]. Endogenous a-oxoaldehyde metabolites are potent glycating agents and react with proteins to form AGEs directly. Important dicarbonyl glycating agents are glyoxal, MG, and 3-deoxyglucosone (3-DG). Further classification of AGEs relates to the mechanism of AGE formation. “Glycoxidation” refers to glycation processes in which oxidation is involved and the AGEs formed thereby are called “glycoxi- dation products” [1]. CML and Ma-carboxymethyl amino acids are glycoxida- tion products. These are often mainly formed by oxidative degradation of fructosamine and hence have the same proteome site-specific coverage as their fructosamine precursor (Figure 8.1b). There are also minor contributions to CML and Ma-carboxymethyl amino acid residue formation from gly- cation of proteins by glycolaldehyde and glyoxal, which may have a different site-specific distribution [49, 50]. The major AGE quantitatively found in physiological systems is the MG-derived hydroimidazolone [18]. MG reacts predominantly with arginine residues to form sequentially a glycosylamine, dihydroxyimidazolidine, and hydroimidazolone MG-H1 residues (Figure 8.1c). Other structural isomers are also found: MG-H1, MG-H2, and MG-H3 [19];

Table 8.1 Components of the fructosamine proteome.

Species

Protein

Hotspot

sites

Extent of modification

Functional

impairment

Reference

Human

Apolipoprotein A1 Apolipoprotein E

Bisphosphoglycerate

mutase

CD59

Complement factor B

Gastric inhibitory polypeptide

Glucagon-like

peptide-1

Hemoglobin a2p2

K239

K93

K158

K41

K266

Y1

1H

a-K61

P-V1

P-K66

4%

Unknown

Unknown

Unknown

Unknown

Unknown

Unknown

5% (аф, 0.6:1)

None

Impairs heparin binding

Inactivation

Inactivation

Increased insulin release

Decreased insulin release

Increased oxygen binding in T state

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28-30]

Human

Insulin

Microglobulin, p2 Serum albumin

Superoxide

dismutase-1

P-F1

I1

D1

K199

K439

K525

K122

K128

Unknown

10%

Unknown

Decreased activity

Aggregation in chronic renal dialysis

Decreased drug binding and leakage through the glomerular filter

Inactivation

[31]

[32]

[33, 34] [35]

Bovine

Crystallin, aA

K11

K78

Unknown

[36]

Bovine

Crystallin, aB

Crystallin, yB

Glutathione

peroxidase-1

Insulin

K90

K92

G1

K2

K117

a-G1

P-F1

P-K29

Unknown

Unknown

Unknown

Unknown

Inactivated

[36]

[37]

[38]

[39]

Table 8.1 (Continued)

Species

Protein

Hotspot

sites

Extent of modification

Functional

impairment

Reference

Major intrinsic peptide

K238

K259

Unknown

Affects membrane permeability

[40]

Bovine

Ribonuclease A Serum albumin

K1

K7

K41

K12

K136

K211

K232

K377

K524

Unknown

10%

[41]

[42]

Rat

Collagen I

Aldo-keto reductase 1A1

a1-K434

a2-K453

a2-K479

a2-K924

K67

K84

K140

  • 50-70%
  • 27-33%
  • 24-29%
  • 22-28%
  • 18%

Increased susceptibility to cross-linking

Inactivation

[43]

[44]

Modifying agent: D-glucose.

isomer MG-H1 is usually dominant in vivo [51]. The half-life for reversal of glycosylamine/dihydroxyimidazolidine formation is ca. 1.8 days and for reversal of hydroimidazolone is ca. 12 days at pH 7.4 and 37 °C [52]. The stability of the hydroimidazolone decreases with increasing pH; the half-life of MG-H1 is 0.87 days at pH 9.4 [19]. Hence dihydroxyimidazolidine and hydroimidazolone residues derived from arginine residues may be detected in mass spectromet- ric analysis of glycated proteins. Glucose-derived Schiff's base and fructosa- mines degrade to form glyoxal, MG, and 3-DG, and so dicarbonyl-derived AGEs may be detected in proteins glycated by glucose too [45, 53].

Glycation of proteins by MG is found at levels of 1-5% in most proteins but increases to ca. 50% in the human lens of elderly where there is limited protein turnover [20, 51]. It often occurs at functional domain of proteins and leads to protein inactivation and dysfunction. This may be because arginine residues have the highest probability (20%) of any amino acid to be found in a functional domain and there is loss of positive charge on the formation of MG-H1 [3]. Gene knockout of glyoxalase 1 (Glo1), the enzyme that protects against glyca- tion by MG, is embryonically lethal, and increased MG concentration, or dicarbonyl stress, imposed by Glo1 deficiency accelerates the aging process and exacerbates diseases - including cardiovascular disease, diabetes, renal failure, and neurological disorders [6]. Proteins susceptible to MG glycation are called the “dicarbonyl proteome” (Table 8.2).

Glycated proteins undergo proteolysis in physiological systems to release glycated amino acids called “glycation-free adducts” These are found in plasma and other body fluids. They are excreted from the body in urine. Urinary excretion of glycation-free adduct increases from 2- to 15-fold in diabetes and renal failure [8, 61]. Glycation-free adducts in ultrafiltrate of physiological fluids are detected and quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) data acquisition mode. In positive ion mode electrospray tandem mass spectrometry, FL dehydrates in the vapor phase and enters the mass analyzer as the singly charged oxonium ion (M+144) [18, 83]. The LC-MS/MS analysis is extended to quantify total glycation adduct contents of purified proteins and protein extracts of cells and extracellular matrix by prior exhaustive enzymatic hydrolysis [18]. Conventional acid hydrolysis cannot be used because of low analytical recoveries [19].

Table 8.2 Components of the dicarbonyl proteome.

Species

Protein

Hotspot

sites

Arg

agent

Extent of modificatioi

Functional n impairment

Reference

Human

Apolipoprotein

A1

Apolipoprotein

B100

CD4 antigen Collagen-IV

R27

R123

R149

R18

R59

a1-R390

a2-R889

a2-R1452

a3-1404

MG

MG

^HOPhG

MG

ca. 1%

Unknown

ca. 5%

R27, increased catabolism; R123, decreased stability; R149, impaired

functional activity

Increased density, proteoglycan binding, and atherogenicity

Binding of HIV pp120

Decreased integrin binding

[12]

[11]

[54]

[55, 56]

Table 8.2 (Continued)

Species

Protein

Hotspot

sites

Arg

agent

Extent of modification

Functional

impairment

Reference

Human

Crystallin, aA

P-Defensin-2

Fibrin(ogen)

R12

R65

R157

R163

R22

R23

a-R167

a-R199

a-R491

a-R528

P-R149

P-R304

MG

Glyoxal/

MG

MG

Unknown

Unknown

Unknown

Increased

chaperone

activity

Decreased

antimicrobial

activity

Abnormal

thrombosis

and

fibrinolysis

[57]

[58]

[59]

Human

Heat shock protein-27

Hemoglobin

a2P2

HIF1a-

coactivator

p300

HIV-1

nucleocapsid

protein

R75

R89

R94

R127

R136

R140

R188

a-R31

a-92

a-141

P-R30

P-R 40

P-R104

R354

R7

R10

R32

MG

MG

MG

Kethoxal

Unknown

ca. 2.6%

Unknown

Unknown

Enhanced protection against oxidative stress

Increased oxygen binding

Decreased

hypoxia

response

Binding of RNA stem-loops 2 and 3

[60]

[61-63]

[64]

[65]

Table 8.2 (Continued)

Species

Protein

Hotspot

sites

Arg

agent

Extent of modification

Functional

impairment

Reference

HLA-DR1

Insulin

IgG

(monoclonal)

Plasminogen

Proteasome, 20S subunits

a-50

a-123

P-189

R46

LC-R30

R504

R530

R561

P2-R85

P4-R224

P4-231

P5-123

P5-128

^HOPhG

MG

MG

MG

MG

Unknown

Unknown

5%

Unknown

Unknown

Surface ligand binding

Aggregation Acidic variant

Likely functional changes to cleavage and Lys binding pocket in fibrinolysis

Decreased

proteasome

activity

[66]

[67]

[68]

[69]

[70]

Bovine

Ribonuclease

A

Seminal plasma protein PDC-109

Serum

albumin

Ubiquitin

R10

R39

R85

R57

R64

R104

R114

R186

R218

R257

R410

R428

R54

R72

R74

Glyoxal/

MG

cHxG

MG

Kethoxal

Unknown Unknown ca. 1%

Unknown

Inhibition

Heparin binding

Inhibition of esterase activity, prostaglandin breakdown, and decreased drug binding

[71, 72] [73] [74, 75]

[65]

Rabbit

Muscle

creatine kinase

R129

R131

R134

PhG

Unknown

Inactivation

[76]

Table 8.2 (Continued)

Species

Protein

Hotspot

sites

Arg

agent

Extent of modification

Functional

impairment

Reference

Mouse

mSin3a

corepressor

R925

MG

Unknown

Increased

angiopoietin-2

activity

[77]

Chicken

Lysozyme

R5

R73

R112

R125

cHxG

Unknown

[78]

Streptomyces

coelicolor

3-

Dehydroquinate

dehydratase

R23

cHxG

Unknown

Inactivation

[79]

Aspergillus

nidulans

3-

Dehydroquinate

dehydratase

R19

cHxG

Unknown

Inactivation

[79]

Aspergillus

sp.

Amadoriase II

R112

R114

pHOPhG

Unknown

Inactivation

[80]

Sorghum

bicolor

Malate

dehydrogenase

R87

R134

R140

R204

cHxG

Unknown

Cofactor binding

Catalytic

mechanism

Substrate binding

Catalytic

mechanism

[81]

Hepatitis C virus

E2 envelope protein

R587

R630

R651

Unknown

Antibody

binding

[82]

Modifying agent key: cHxG, cyclohexanedione; MG, methylglyoxal; pHOPhG,

p-hydroxyphenylglyoxal; PhG, phenylglyoxal. Other: glyoxal and kethoxal. NB: These are all a-oxoaldehydes, but only glyoxal and Mg are physiological glycating agents. Reactivity of sites to other agents is assumed to indicate reactivity of the arginine residue site to dicarbonyl glycation.

 
Source
Found a mistake? Please highlight the word and press Shift + Enter  
< Prev   CONTENTS   Next >
 
Subjects
Accounting
Business & Finance
Communication
Computer Science
Economics
Education
Engineering
Environment
Geography
Health
History
Language & Literature
Law
Management
Marketing
Mathematics
Political science
Philosophy
Psychology
Religion
Sociology
Travel