Accepted Manuscript

Title: Activation of MMP-9 activity by acrolein in saliva from

patients with primary Sjögren’s syndrome and its mechanism

Authors: Takeshi Uemura, Takehiro Suzuki, Ryotaro Saiki,

Naoshi Dohmae, Satoshi Ito, Hoyu Takahashi, Toshihiko
Toida, Keiko Kashiwagi, Kazuei Igarashi

PII: S1357-2725(17)30094-8
DOI: http://dx.doi.org/doi:10.1016/j.biocel.2017.05.004
Reference: BC 5120

To appear in: The International Journal of Biochemistry & Cell Biology

Received date: 17-12-2016

Revised date: 18-3-2017
Accepted date: 4-5-2017

Please cite this article as: Uemura, Takeshi., Suzuki, Takehiro., Saiki, Ryotaro., Dohmae,
Naoshi., Ito, Satoshi., Takahashi, Hoyu., Toida, Toshihiko., Kashiwagi, Keiko., &
Igarashi, Kazuei., Activation of MMP-9 activity by acrolein in saliva from patients with
primary Sjögren’s syndrome and its mechanism.International Journal of Biochemistry
and Cell Biology http://dx.doi.org/10.1016/j.biocel.2017.05.004

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Highlights

MMP-9 activity in saliva of primary Sjögren’s syndrome (pSS) patients was examined.

The specific activity of MMP-9 in saliva was elevated about 2.4-fold in pSS patients.

The increase in MMP-9 activity was due to acrolein-conjugation with Cys99.

Acrolein-conjugation at Cys99 caused the active site of MMP-9 to be exposed.

Activated 82 and 68 kDa MMP-9s were not detected in saliva of pSS patients.

1
Activation of MMP-9 activity by acrolein in saliva from patients with primary Sjögren’s

syndrome and its mechanism

Takeshi Uemuraa, Takehiro Suzukib, Ryotaro Saikia, Naoshi Dohmaeb, Satoshi Itoc,d, Hoyu

Takahashid, Toshihiko Toidae, Keiko Kashiwagif, and Kazuei Igarashia,e,*

a
Amine Pharma Research Institute, Innovation Plaza at Chiba University, 1-8-15 Inohana,

Chuo-ku, Chiba, Chiba 260-0856, Japan

b
RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198,

Japan
c
Department of Rheumatology, Niigata Rheumatic Center, 1-2-8 Hon-cho, Shibata, Niigata

957-0054, Japan
d
Niigata Prefectural Kamo Hospital, 1-9-1 Aomi-cho, Kamo, Niigata 959-1397, Japan
e
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku,

Chiba, Chiba 260-8675, Japan

f
Faculty of Pharmacy, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025,

Japan

*Corresponding author at: Amine Pharma Research Institute, Innovation Plaza at

Chiba University, 1-8-15 Inohana, Chuo-ku, Chiba, Chiba 260-0856, Japan. Phone,

+81-43-224-7500; Fax: +81-43-379-1050.

E-mail address: iga16077@faculty.chiba-u.jp (K. Igarashi).

Abbreviations: APMA, 4-aminophenylmercuric acetate; MMP-9, matrix

metalloproteinase-9; PC-Acro, protein-conjugated acrolein; pSS, primary Sjögren’s

syndrome.

2
ABSTRACT

We have recently reported that the altered recognition patterns of immunoglobulins due to

acrolein conjugation are at least partially responsible for autoimmune diseases in patients with

primary Sjögren’s syndrome (pSS). In the current study, it was found that the specific

activity (activity/ng protein) of metalloproteinase-9 (MMP-9) in saliva was elevated about

2.4-fold in pSS patients. Accordingly, it was examined whether MMP-9 is activated by

acrolein. It was found that the MMP-9 with 92 kDa molecular weight was activated by

acrolein. Under the conditions studied, Cys99, located in the propeptide, was conjugated

with acrolein together with Cys230, 244, 302, 314, 329, 347, 361, 373, 388 and 516, which

are located in fibronectin repeats and glycosyl domains, but not on the active site of MMP-9.

In addition, 82 and 68 kDa constructs of MMP-9s, lacking the NH2-terminal domain that

contains Cys99, were not activated by acrolein. The results suggest that acrolein

conjugation at Cys99 caused the active site of MMP-9 to be exposed. Activation of MMP-9

by acrolein was inhibited by cysteine, and slightly by lysine, because these amino acids

inhibited acrolein conjugation with MMP-9. Conversely, MMP-9 activity in the presence of

50 μM acrolein was enhanced by 100 μM histidine. This was due to the inhibition of

acrolein conjugation with His405 and 411 located at the Zn2+ binding site of MMP-9. These

results suggest that activation of 92 kDa MMP-9 by acrolein is involved in tissue damage in

pSS patients and is regulated by cysteine and histidine, and slightly by lysine. Activated 82

and 68 kDa MMP-9s were not detected in saliva of pSS patients by Western blotting.

Keywords: Acrolein conjugation; Activation of 92 kDa MMP-9 (matrix metalloproteinase-9);

Cysteine switch; Propeptide domain; Primary Sjögren’s syndrome.

3
1. Introduction

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder mainly

affecting the salivary and lacrimal glands and is characterized by dry mouth and eyes as a

result of decreased salivary and lacrimal secretion caused by destruction of these glands

(Konttinen et al., 1994; Moutsopoulos et al., 1979). Autoantibodies recognizing Sjögren’s

syndrome A (SSA, Ro) and Sjögren’s syndrome B (SSB, La) proteins were frequently found

in sera of pSS patients (Franceschini and Cavazzana, 2005; Goeb et al., 2007), and we

recently found that the activities of autoantibodies recognizing SSA (Ro) and SSB (La)

proteins in saliva were approximately 3- to 5-fold higher than those from control subjects

(Hirose et al., 2015). Matrix metalloproteases (MMPs), particularly gellatinases (MMP-2

and MMP-9), were also involved in tissue damage of pSS patients (Hanemaaijer et al., 1998;

Perez et al., 2000; Ram et al., 2006). Oxidative stress, which can lead to various pathologies,

is thought to be caused by two kinds of compounds – first, reactive oxygen species (ROS)

such as superoxide anion radical (O2•‾), hydrogen peroxide (H2O2) and hydroxyl radical

(•OH), and second, unsaturated aldehydes such as acrolein (CH2=CH-CHO) and

4-hydroxynonenal (Hensley et al., 2000; Kehrer and Biswal, 2000). We previously reported

that acrolein, which is mainly produced from polyamines, especially from spermine, is

strongly involved in tissue damage associated with chronic renal failure and brain infarction

(Igarashi et al., 2006; Saiki et al., 2009; Sakata et al., 2003; Tomitori et al., 2005). We found

that the toxicity of acrolein is more pronounced than that of ROS (Sharmin et al., 2001;

Yoshida et al., 2009). Hence, we determined whether acrolein is involved in the destruction

of salivary glands in pSS patients, and found that protein-conjugated acrolein (PC-Acro) in

saliva is well correlated with the destruction of salivary glands in pSS patients (Higashi et al.,

2010). Actually, an autoimmune disorder was partially due to the acrolein conjugation with

immunoglobulins (Hirose et al., 2015).

4
 It has been reported that the activity of MMP-9 is enhanced by acrolein mainly at the

transcriptional level in mouse lung (Deshmukh et al., 2008) and in human macrophages

(O'Toole et al., 2009). However, the mechanism of acrolein activation of MMP-9 was not

studied in detail thus far. In this study, we determined whether the activity of MMP-9,

derived from saliva or using purified MMP-9, is enhanced by acrolein. Our results indicate

that the activity of MMP-9 is enhanced by acrolein-conjugation at Cys99 located at the

NH2-terminal end, i.e. the propeptide domain, of MMP-9, which caused the active site of

MMP-9 to be exposed. The activation of MMP-9 by acrolein was similar to the activation

by S-nitrosylation at Cys99 (Gu et al., 2002).

2. Materials and methods

2.1. Subjects and collection of saliva and blood

We examined 11 women with pSS (68.8 ± 9.4 years) and 10 women with dry eye

and/or dry mouth (72.0 ± 6.8 years) as control, who did not fulfill classification criteria of pSS

proposed by the Japanese Study Group on Diagnostic Criteria for Sjögren’s syndrome (Table

1) (Deshmukh et al., 2008; O'Toole et al., 2009). None of the control subjects had

significant anti-SSA (Ro) and anti-SSB (La) antibodies, but 2 of them were Gum test positive

and 2 of them were Schirmer test positive. Keratoconjunctivitis sicca was observed in one

subject. Informed consent was given by each participant, and our study protocol was

approved by the ethics committees of the Graduate School of Pharmaceutical Sciences, Chiba

University, and of the Niigata Prefectural Kamo Hospital. Experiments were conducted in

accordance with the Declaration of Helsinki Principles. Patients and control subjects

underwent the same dental evaluation, procedures, in the same order, between 9:00 and 12:00

a.m. at the Niigata Prefectural Kamo Hospital. Interviews, oral clinical examinations, and

collection of saliva and blood were performed by the same doctor. Whole-mixed saliva was

5
collected during stimulation by chewing paraffin for 10 min. Immediately after collection,

the saliva samples were centrifuged at 10,000 x g for 5 min at 4 °C and the supernatants were

frozen at -80 °C until use. Flow rate of saliva (ml/min) is defined as a milliliter of saliva

collected in 10 min divided by 10. Average flow rate and protein concentration of saliva of

control subjects and pSS patients were 1.0 and 0.3 ml/min, and 2.1 and 3.7 mg /ml,

respectively. Blood containing 3 U/ml heparin was centrifuged at 1,500 x g for 10 min at

4 °C, and plasma was carefully collected to avoid contamination by erythrocytes.

2.2. Materials

Human purified recombinant 92 kDa MMP-9 proenzyme was purchased from

Calbiochem, CA. Cysteine, lysine and histidine were obtained from Nacalai Tesque, Japan,

and 4-aminophenylmercuric acetate (APMA) was from Sigma-Aldrich, MO. Acrolein was

purchased from Tokyo Chemical Industry, Japan.

2.3. Measurement of MMP-9 activity

MMP-9 activity in saliva was measured by the method of Hanemaaijer et al

(Hanemaaijer et al., 1999) using urokinase as substrate in the MMP-9 assay system, GE

Healthcare, UK. Incubation was performed at 37 ºC for 30 min. APMA treatment of

MMP-9 was carried out as described by Watanabe et al. (Watanabe et al., 1993). In brief, 1

µg of 92 kDa MMP-9 was incubated with 1 mM APMA in the assay buffer (0.4 ml)

containing 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5 mM CaCl2, 1 µM ZnCl2 and 0.01%

BrijTM 35 (Sigma-Aldrich) at 37 ºC for 24 h. After incubation, APMA was removed by

filtering through Amicon Ultra 10K filter (Merck Millipore, Germany) three times with

phosphate buffered saline (PBS). For acrolein treatment of MMP-9, saliva (0.1 ml) or 100

ng 92 kDa MMP-9 in PBS (0.02 ml) was incubated with 20 - 500 µM acrolein at 37 ºC for 3 h.

Acrolein was also removed like APMA as described above. The activity of purified 92 kDa

6
MMP-9 and APMA-treated MMP-9 was measured at 37 ºC for 30 min as described by

Johnson et al (Johnson et al., 2007) using 5 ng protein and SonsoLyte®520 MMP-9 Assay Kit,

ANASPEC, CA, in which the substrate for MMP-9 is

QXL®520-PLGC(Me)HAR(D-)K(5-FAM)-NH2.

2.4. Western blotting

Twenty µg of saliva protein or 100 ng of MMP-9 protein was separated on the 10%

SDS-polyacrylamide gel and transferred to PVDF membrane (GE Healthcare). MMP-9 and

acrolein conjugated MMP-9 were detected using anti-MMP-9 antibody (Cell Signaling

Technology) or polyclonal antibody against protein-conjugated acrolein (MoBiTec, Germany)

and ECL Western blotting detection system, GE Healthcare. The band intensity was

measured using NIH ImageJ 1.49v program (Schneider et al., 2012). Proteins were stained

with Coomassie Brilliant Blue-R250 (CBB).

2.5. Immunoprecipitation

For evaluation of acrolein-conjugated MMP-9 levels in saliva, immunoprecipitation

was performed as described previously (Uemura et al., 2008). In brief, 0.1 ml of saliva of

control subjects and pSS patients was incubated with anti-MMP-9 antibody in IP buffer

containing 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% NP-40 at 4 ºC for 16 h with gentle

rotation. Saliva samples were then incubated with protein A agarose beads (GE Healthcare)

at 4 ºC for 16 h. After washing 5 times with 1 ml of IP buffer, beads were suspended with 40

l of SDS-PAGE sample buffer and boiled for 5 min. Beads associated proteins were

separated on a 10% SDS-polyacrylamide gel, transferred to PVDF (GE Healthcare) and

MMP-9 and acrolein-conjugated MMP-9 were detected as described above.

2.6. Mass spectrometry

7
 This was performed as described previously (Cai et al., 2009; Yoshida et al., 2010).

A protein band of MMP-9 obtained with SDS-polyacrylamide gel electrophoresis was excised,

reduced with dithiothreitol and alkylated by acrylamide. The protein in-gel was digested

with either trypsin (TPCK-treated, Worthington Biochem. Co.) or endoproteinase Asp-N

(Roche Applied Science, USA) at 37 °C overnight. An aliquot of digestion mixture was

separated using nano-electrospray ionization spray column (NTCC analytical column, C18,

φ75 μm × 100 mm, 3 μm, Nikkyo Technos Co., Ltd., Tokyo, Japan) at a flow rate 300 nl/min

and subjected on-line to a Q Exactive (Thermo Fisher Scientific, USA) with a nanospray ion

source. MS and MS/MS data were acquired in a data-dependent TOP10 method. Obtained

MS/MS data were searched against the SwissProt 2015_08 database or in-house database with

Mascot Version 2.5 (Matrix Science) using the following parameters: Taxonomy, Homo

sapiens (human) (20204 sequences); Type of search, MS/MS Ion Search; Enzyme, Trypsin

and/or Asp-N-ambic; Fixed modification, none; Variable modifications : Deamidated (NQ),

Oxidation (M), Propionamide (C), Gln->pyro-Glu (N-term Q), MP-lysine (K), FDP-lysine

(K), Nim-propanalhistidine (H), Acrolein adduct (C), Acrolein adduct2 (N-term) ; Mass

values, Monoisotopic ; Peptide Mass Tolerance : ± 15 ppm ; Fragment Mass Tolerance: ± 20

mmu ; Max Missed Cleavages : 3 ; Instrument type : ESI-TRAP.

2.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism version 6.0d for Mac,

GraphPad Software, La Jolla California USA, www.graphpad.com. Differences between

two groups were compared using Student’s t-test. For comparison of multiple groups,

one-way ANOVA followed by Dunnett’s multiple comparisons test was used.

3. Results

8
3.1. Increase in specific activity of MMP-9 in saliva from pSS patients

We first determined whether MMP-9 in saliva of pSS patients is activated by acrolein

as reported in mouse lung (Deshmukh et al., 2008) and in human macrophages (O'Toole et al.,

2009). The level of 92 kDa MMP-9 measured by ELISA in saliva of pSS patients was

slightly higher (about 1.4-fold) than that in saliva of control subjects (Fig. 1A). Activated 82

and 68 kDa MMP-9s (Rosenblum et al., 2007; Vandooren et al., 2013) were not detected in

saliva by Western blotting with both control subjects and pSS patients. This was confirmed

even if 5-times more proteins in saliva were used for Western blotting (data not shown).

However, the specific activity of MMP-9 in saliva of pSS patients was significantly higher

(about 2.4-fold) than that in saliva of control subjects (Fig. 1B), consistent with the idea that

MMP-9 in pSS patients is activated by acrolein. Indeed, the level of acrolein-conjugated

MMP-9 in saliva was higher in pSS patients than control subjects (Fig. 1C), and

concentration-dependent activation of MMP-9 by acrolein was confirmed using saliva in

control subjects treated with 20 to 500 μM acrolein at 37 ºC for 3 h (Fig. 1D). Since acrolein

concentration in saliva is less than 20 μM (data not shown), 2.5-fold increase in MMP-9

activity in saliva of pSS patients is reasonable.

Using purified MMP-9 treated with 20 μM acrolein at 37 ºC for 3 h,

acrolein-conjugated amino acid residues were identified. MMP-9 (molecular mass, 92 kDa,

707 amino acid residues) consists of propeptide, active site, fibronectin type II repeats, Zn2+

binding domain, glycosyl and hemopexin domains (Fig. 2A) (Vandooren et al., 2013). The

catalytic domain consists of active site and Zn2+ binding domain (Nagase and Woessner, 1999;

Ra and Parks, 2007). Acrolein-conjugated amino acids were identified by LC-MS/MS.

Eleven cysteine residues (Cys99 in propeptide, Cys230, Cys244, Cys302, Cys314, Cys327,

Cys347, Cys361, Cys373 and Cys388 in fibronectin type II repeats, and Cys516 in glycosyl

9
domain), two histidine residues (His405 and His411 in Zn2+ binding domain) and two lysine

residues (Lys384 in fibronectin type II repeats and Lys535 in hemopexin domain) were found

to be acrolein-conjugated (Fig. 2A and 2B). Examples of MS/MS spectra of peptides having

acrolein conjugated cysteine are shown in peptides 99-CGVPDLGR-106 and

222-FGNADGAACHFPFIFEGR-239 of MMP-9 (Fig. 2C). Similar results were obtained

with purified MMP-9 treated with 100 μM acrolein (data not shown).

The effect of acrolein on MMP-9 activity was then studied using purified 92 kDa

MMP-9 and activated MMP-9s, consisting of both 82 kDa and 68 kDa MMP-9s. Activated

MMP-9s were prepared by treatment of 92 kDa MMP-9 with APMA (4-aminophenylmercuric

acetate) (Watanabe et al., 1993). As shown in Fig. 3A, the increase in purified MMP-9

activity by acrolein was nearly equal to that in saliva MMP-9. Under these conditions,

conversion of 92 kDa MMP-9 to 82 kDa and 68 kDa MMPs was not observed (Fig. 3B).

The activity of 82 kDa and 68 kDa MMP-9s was increased approximately 10-fold without

acrolein treatment compared with the activity of 92 kDa MMP-9, but it was not activated by

acrolein (Fig. 3A). Treatment with high concentration of acrolein (500 μM) inhibited

MMP-9 activity of 82 kDa and 68 kDa MMP-9s. In addition, degree of acrolein conjugation

with 82 kDa MMP-9 was weaker compared with 92 kDa MMP-9, judging from the relative

amount of acrolein-conjugated MMP-9 (PC-Acro) (Fig. 3B). The 68 kDa MMP-9, lacking

both propeptide and hemopexin domains, was not conjugated with acrolein (Fig. 3B),

suggesting that the hemopexin domain influences the structure of the catalytic domain of

MMP-9. Since the activity of 82 kDa MMP-9, lacking the propeptide domain, was not

influenced significantly by acrolein (Fig. 3A and 3B), the results strongly suggest that Cys99

located in the propeptide domain is involved in acrolein stimulation of MMP-9 activity. This

is the first report that cysteine 99 in the propeptide domain is involved in the enhancement of

MMP-9 activity through acrolein conjugation. Our results are consistent with an idea that a

10
disengagement of the propeptide from the active center of MMP-9 causes its activation

(Bannikov et al., 2002).

3.2. Effect of Cys, Lys and His on MMP-9 activity

Since acrolein interacts with Cys most strongly, but also with Lys and His (Hirose et

al., 2015), and it conjugated with Cys, Lys and His residues in MMP-9 (Fig. 2A), the effects

of these amino acids on MMP-9 activity were examined. As shown in Fig. 4A, more than 10

μM Cys and Lys reduced MMP-9 activity in the presence of 50 μM acrolein, suggesting that

Cys and Lys interrupt acrolein conjugation with Cys99 on MMP-9. It was also found that

acrolein conjugation with MMP-9 was strongly inhibited by Cys and weakly by Lys (Fig. 4B).

The relative amount of acrolein-conjugated MMP-9 in the presence of 1 mM Cys or 1 mM

Lys was 4 or 50% compared with that in the absence of amino acid. Conversely, His at

concentrations of 100 to 1000 μM stimulated MMP-9 activity (Fig. 4A), and conjugation of

acrolein with Cys residues in MMP-9 was not strongly inhibited by 1 mM His (Fig. 4B).

The results suggest that conjugation of acrolein with His405 and His411 located in the active

site of MMP-9 (Fig. 2A) was reduced by high concentrations of His. His405 and His411

have been shown to constitute the Zn2+ binding site together with His401 (Elkins et al., 2002).

The addition of 1 mM EDTA to the assay mixture inhibited MMP-9 activity completely (Fig.

4A), confirming that Zn2+ is essential for MMP-9 activity.

The mechanism of the activation of MMP-9 by acrolein and His was then studied by

Lineweaver-Burk plot analysis. Although the kcat value increased, the Km value did not

change when MMP-9 was treated with both acrolein and His (Fig. 5). The kcat value

increased from 0.34 to 7.34/min. The results suggest that a structural change through Cys99

to Cys99-Acro and His405 & 411-Acro to His405 & 411 would increase the interaction of the

Zn2+ binding domain with the active site of MMP-9 preceding fibronectin repeats (Fig. 2A).

11
4. Discussion

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder characterized

by destruction of some secretory glands. The mechanism can be at least partly explained by

the involvement of acrolein. Antigen recognition was modified through acrolein conjugation

with several amino acid residues in immunoglobulins, in particular acrolein conjugation with

residues in the variable regions of immunoglobulins (Hirose et al., 2015). These modified

immunoglobulins recognized protein(s) expressed in cells or tissues such as SSA (Ro) and

SSB (La) (Hirose et al., 2015).

With regard to activation of MMP-9 by acrolein, it is important to know its

mechanism because MMP-9 is involved in cerebrovascular damage in acute ischemic stroke

(Castellanos et al., 2003; Kelly et al., 2008) and in diabetes (Navaratna et al., 2013) as well as

autoimmune diseases. It has been reported that activation of MMP-9 by acrolein mainly

occurs at the level of transcription in mouse lung (Deshmukh et al., 2008) and in human

macrophages (O'Toole et al., 2009). However, our results indicate that activation of MMP-9

also occurs at the level of acrolein-conjugation with Cys99 in the propeptide domain. In the

case of a related protease, MMP-7, a highly conserved domain called the cysteine switch has

been proposed to regulate MMP-7 activity (Fu et al., 2001). Hypochlorous acid (HOCl)

produced from H2O2 by myeloperoxidase interacts with a cysteine residue in the propeptide

domain, and causes the cysteine switch to sulfinic acid. This causes autolytic cleavage of

MMP-7, Zn2+ is able to interact with the catalytic domain, and MMP-7 is activated. In this

study, we found that activation of MMP-9 by acrolein occurred through acrolein conjugation

with Cys99. Thus, similarly to the cysteine switch in MMP-7, an interaction between Cys99

and Zn2+ is disturbed through acrolein-conjugation with Cys99, and Zn2+ may be able to

12
function as a co-activator of MMP-9 through interaction with the active site. A model for

the activation of MMP-9 by acrolein is shown in Fig. 6. His405 and His411 at the active site

were also conjugated with acrolein, but the degree of acrolein conjugation was weaker than

that with Cys99 (data not shown). Thus, significant inhibition of MMP-9 through acrolein

conjugation with His405 and His411 was not observed in saliva from pSS patients.

It has been reported that S-nitrosylation of MMP-9, similar to acrolein conjugation,

causes the cysteine switch and activation of MMP-9 (Gu et al., 2002). We recently reported

that acrolein conjugation with Cys150 of glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) inhibits its activity (Nakamura et al., 2013) similar to S-nitrosylation of the same

Cys residue (Sen et al., 2008). Cysteine-modified GAPDH shifted to nuclei and caused

apoptosis (Nakamura et al., 2013; Sen et al., 2008). Thus, acrolein modification of cysteine

residues in enzymes may lead to either activation as seen in MMP-9 or inhibition as seen in

GAPDH. Generally, acrolein causes inactivation of enzymes or proteins such as ADP/ATP

translocase 1 (Han et al., 2007), actin (Dalle-Donne et al., 2007), NF-B (Lambert et al.,

2007) and protein tyrosine phosphatase 1B (Seiner et al., 2007). Activation of MMP-9 by

acrolein is a rare modification, but likely plays a key role in acrolein toxicity together with

inactivation of several other enzymes or proteins.

Acknowledgments

We thank Drs. A. J. Michael and K. Williams for their help in preparing the manuscript.

Conflict of interest

The authors declare that they have no conflicts of interest.

13
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Figure legends

Fig. 1. Increase in MMP-9 activity in saliva of pSS patients. Amount (A) and activity (B)

of MMP-9 in saliva from 10 control subjects and 11 pSS patients were measured as described

in Materials and methods. B. Blank value without incubation (0.003/ng MMP-9) was

subtracted from MMP-9 specific activity. C. The level of MMP-9 and acrolein-conjugated

MMP-9 in saliva of control subjects and pSS patients were evaluated by Western blotting after

immunoprecipitation. D. Effect of acrolein on MMP-9 activity was evaluated by incubation

of saliva from control subjects with 20 - 500 μM acrolein at 37 ºC for 3 h. ns, p ≥ 0.05; * p

< 0.05; ** p < 0.01; *** p < 0.001.

Fig. 2. Acrolein conjugated amino acid residues in human purified MMP-9 treated with 20

µM acrolein at 37 ºC for 3 h. A. Fifteen kinds of the identified acrolein-conjugated amino

acid residues are shown on the simplified structure of MMP-9. Propeptide, active,

fibronectin repeats, Zn2+ binding site (active), glycosyl and hemopexin domains were

indicated together with the molecular weight. B. Underlined peptides were identified by

LC-MS/MS. Acrolein-conjugated amino acid residues were shown in red. Arrow indicates

the point, at which the signal peptide is released. C. MS/MS spectra of two fragments of

MMP-9 were shown. y* indicates y-NH3. Cys99 and Cys230 were conjugated with

acrolein by Michael addition and Schiff base was formed intramolecularly at the N-terminus

of the peptide (Bradley et al., 2010).

Fig. 3. Effect of acrolein on MMP-9 and APMA treated MMP-9. A. Treatment of MMP-9

with APMA and measurement of MMP-9 activity in the presence of various concentrations of

acrolein were performed as described in Materials and methods. ** p < 0.01; *** p < 0.001.

B. Levels of MMP-9 and acrolein conjugated MMP-9 were measured by CBB staining and

Western blotting using antibody against PC-Acro, respectively. The relative amount of

20
acrolein-conjugated MMP-9 was measured using NIH Image J 1.49v program (Schneider et

al., 2012).

Fig. 4. Effect of Cys, Lys and His on MMP-9 activity. A. Purified MMP-9 was treated

with 50 µM acrolein in the presence of various concentrations of Cys, Lys and His at 37 ºC

for 3 h, and the activity was measured as described in Materials and methods. Where

indicated, 1 mM EDTA was added together with 50 μM acroelin. B. Levels of

acrolein-conjugated MMP-9 treated with 1 mM Cys, Lys and His were determined by CBB

staining and Western blotting using antibody against PC-Acro. The relative amount of

acrolein-conjugated MMP-9 was measured using NIH ImageJ 1.49v program (Schneider et al.,

2012).

Fig. 5. Determination of the Km and kcat values of acrolein-activated MMP-9 in the presence

and absence of His. The substrate for MMP-9 was shown in Materials and methods.

MMP-9 was treated with 50 μM acrolein in the presence and absence of 100 μM His at 37 ºC

for 3 h, and the activity was measured by changing the concentration of the substrate. The

Km and kcat values were determined by Lineweaver-Burk plot. ns, p ≥ 0.05; ** p < 0.01; ***

p < 0.001 vs value of no addition.

Fig. 6. Model of acrolein activation of MMP-9. Green, propeptide domain; blue,

fibronectin repeats; orange, active site; brown, glycosyl domain; red, hemopexin domain.

Catalytic site, including Zn2+ binding site (see Fig. 3) is shown in orange.

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Table 1. Characteristics of control subjects and pSS patients

Control pSS
Number 10 11
Age (years) 72.0 ± 6.8 68.8 ± 9.4
Anti-SSA antibody positive (plasma) 0 9
Anti-SSB antibody positive (plasma) 0 4
Gum test positive 2 9
Schirmer test positive 2 9
Keratoconjunctivitis Sicca 1 9

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