The autoantigen Pso p27 – a posttranslational modification of

SCCA molecules

Ole-Jan Iversen, Hilde Lysvand and Lars Hagena , Department of Laboratory Medicine

Children’s and Women’s Health and Department of Cancer research and Molecular Medicinea,

Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

Corresponding author:

Prof. Ole-Jan Iversen

Norwegian University of Science and Technology, Faculty of Medicine

Department of Laboratory Medicine, Children’s and Women’s Health,

Postbox 8905

N-7491 Trondheim

Phone: +47 72573066

Fax: +47 72576416

email: oleji@ntnu.no

Running title: The Autoantigen Pso p27

Key words: Psoriasis, Pso p27, Autoantigen, SCCA

INTRODUCTION

Psoriasis is a chronic inflammatory skin disease which afflicts about 2% of the

population. The significance of immune reactions in the pathogenesis of psoriasis has

been verified through several studies as recently reviewed by Bowcock and Krueger [1]

and Lowes et al. [2]. Family studies have clearly shown the presence of inherited

predisposition of psoriasis [3-5] while twin studies demonstrate the significance of

environmental factors [6-8].

The infiltration of inflammatory cells as an initial event in the development of new skin

lesions suggests that immune reactions play a crucial role in the pathogenesis of

psoriasis [9].

There are two characteristic features of psoriasis which remain to be clarified.

1. The limited skin lesions – suggesting a local production of antigens

responsible for the immune reactions.

2. The permanence of the skin lesions - suggesting antigen variability and

failure with respect to development of immunological tolerance.

The psoriasis associated antigen, Pso p27, is to our knowledge the only antigen in

psoriatic lesions recognized by antibodies obtained from psoriatic scale [10].

The Pso p27 antigen is primarily found in mast cells in psoriatic lesions and is not

present in uninvolved psoriatic skin or skin biopsies from healthy controls [11].

Suppression of Pso p27 antigen is found to coincide with the remission of disease

activity both spontaneously and as a consequence of treatment [11-13]. In this way Pso

p27 fulfil the criteria as a candidate for a localised causal antigen.

2

In this study we perform a protein sequencing of Pso p27 and argue for the suggestion

that Pso p27 is a posttranslational modification of various representatives of the SCCA

family. The protein heterogeneity and antigenic variability is demonstrated through 2-

dimensional gel-electrophoresis and immunoblot using specific monoclonal antibodies.

This heterogeneity and the structural deviations from the native SCCA molecules may

contribute to the failure of immunologic tolerance.

3

MATRIALS AND METHODS

Purification of Pso 27 Protein from Psoriatic Scale

Scale (150 mg) was homogenized in 4 ml 0.1 M Na-carbonatebuffer pH 10.8 containing

0.5 M NaCl by using an UltraTurrax T25 (Rose Scientific Ltd.).The crude extract was

centrifuged at 16.000 x g and immunoglobulins were removed from supernatant by

filtering through Nanosep 100K Omega (Pall Corporation). The extract was neutralized

with 0.1 M HCl and applied on CnBr-act Sepharose 4B (Amersham Biosciences)

coupled with monoclonal anti-Pso p27 antibodies. After washing with PBS the protein

was eluted with 0.1 M Glycin-HCl pH 2,6 containing 0.5 M NaCl.

Sample Preparation for MALDI Analysis

Purified Pso p27 protein was run on a 10 % NuPage Novex Bis-Tris acrylamid gel

(Invitrogen Life Science) using MOPS running buffer. The gel was further stained with

Simply Blue Safe Stain (Invitrogen Life Science). The proteinband containing about 1

µg protein was excised and in-gel digested [14] with Sequence Grade Modified Trypsin

(Promega corp), Endoproteinase Lys-C (Sigma) or Endoproteinase Glu-C (New

England Biolabs). The peptides were further extracted from the gel and desalted using

Stage Tip Purification [15]. The purified material was mixed with an equal volume of 10

mg/ml 2,5-dihydroxybenzoic acid (DHB), air dried on a stainless steel sample stage and

analysed on a Ultraflex III TOF/TOF (Bruker Daltonics) mass spectrometer.

2D Gel-electrophoresis and Immunoblot

4

For 2D PAGE the Pso p27 protein was focused in an immobilized pH gradient pH 4-7

using an IPGphor II unit (GE Healthcare). In the second dimension the protein was

separated in a 12% polyacrylamide gel using the Hoefer SE600 unit (GE Healthcare).

Finally the gel was stained using the Proteosilver stain kit (SIGMA), or transferred to

nitrocellulose for detection of Pso p27 antigen. The nitrocellulose membrane was

blocked with 5% fat-free dry milk in PBS-T followed by incubation with either of the

monoclonal antibodies against Pso p27; 3A3D10 and 2C7D10 [16]. The binding of the

monoclonal antibodies was visualized by incubation with Horseradish persoxidase

conjugated rabbit anti-mouse immunoglobulines (DAKO) and SuperSignal West Femto

(Thermo scientific) as substrate.

5

RESULTS

MS Spectrum and MS/MS Analysis

The MS spectrum of the tryptic digest of Pso p27 is shown in figure 1a. The peptide

mass fingerprint analysis using the Mascot software (Matrix Science) showed good

correlations to SCCA molecules.

MALDI-MS/MS analysis of the various fragments confirmed the relationship between

Pso p27 and SCCA. Furthermore, the sequencing also demonstrated the presence of

various SCCA molecules as for example peak 1871 and 1887 (fig 1b) which revealed

QYTSFHFASLEDVQAK and QYTSFHFALLEDVQAK, respectively, suggesting the

presence of both SCCA1 and SCCA2 molecules.

6

849.464

2177.964

1181.542

1742.862

1022.608 1434.660

1870.877

2306.0731562.752

2100.111 2414.2922599.4341337.645

0.0

0.5

1.0

1.5

2.0

4x10In

tens

. [a.u

.]

800 1000 1200 1400 1600 1800 2000 2200 2400 2600m/z

Figure 1a. MS-spectrum of trypsin digested Pso p27

7

1742.862

1790.8351870.877

1886.905

1913.984

1759.900

1853.8341927.892

0.00

0.25

0.50

0.75

1.00

1.25

1.50

4x10In

tens

. [a.u

.]

1700 1750 1800 1850 1900 1950 2000m/z

Figure 1b. MS-spectrum of trypsin digested of Pso p27 – selected area

8

Peptide Sequences in Pso p27.

MALDI-MS/MS analysis of the m/z = 1689 fragment of the Lys-C digest showed

HVDRSGNVHHQFQK. No peptide fragments between this sequence and the N-

terminal ends of the SCCA molecules were detected (Fig. 2a, b). This indicates that

HVDRSGNVHHQFQK represent the N-terminal end of the Pso p27 antigen.

MALDI-MS/MS analysis of the m/z =1758 fragment of the trypsic digest showed the

sequence AFVEVTEEGVEAAAATAV. No peptide fragments between this sequence

and the C-terminal end of any SCCA molecules were detected. Based on this it seems

reasonable to suggesting that AFVEVTEEGVEAAAATAV represent the C-terminal end

of the Pso p27 antigen.

The results obtained using MALDI-MS/MS after digestion with the three

endoproteases; trypsin, Glu-C and Lys-C showed significant sequence homology with

both SCCA1 and SCCA2 as shown in fig. 2 a and b.

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Pso p27

HVDRSGN

SCCA1 1

MNSLSEANTKFMFDLFQQFRKSKENNIFYSPISITSALGMVLLGAKDNTAQQIKKVLHFDQVTENTTGKAATYHVDRSGN

Pso p27 VHHQFQKLLTEFNKSTDAYELKIANKLFGEKTYLFLQEYLDAIK FYQTSVESVDFANAPEESR

INSWVESQTNEK

SCCA1 81

VHHQFQKLLTEFNKSTDAYELKIANKLFGEKTYLFLQEYLDAIKKFYQTSVESVDFANAPEESRKKINSWVESQTNEKIK

Pso p27

QYTSFHFASLEDVQAKVLEIPYK D

SCCA1 161

NLIPEGNIGSNTTLVLVNAIYFKGRWEKKFNKEDTKEEKFWSNKNTYKSIQMMRQYTSFHFASLEDVQAKVLEIPYKGKD

Pso p27 LSMIVLLPNEIDGLQK LMEWTSLQNMRETRVDLHLPRFKVEESYDLKDTLR

SCCA1 241

LSMIVLLPNEIDGLQKLEEKLTAEKLMEWTSLQNMRETRVDLHLPRFKVEESYDLKDTLRTMGMVDIFNGDADLSGMTGS

Pso p27 GLVLSGVLHKAFVEVTEEGAEAAAATAV

SCCA1 321 RGLVLSGVLHKAFVEVTEEGAEAAAATAVVAFGSSPTSTNEEFHCNHPFLFFIRQNKTNSILFYGRFSSP

Figure 2a. Peptide sequences detected in the enzyme digests of Pso p27 corresponding to SCCA1.

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Pso p27

HVDRSGN

SCCA2 1

MNSLSEANTKFMFGLFQQFRKSKENNIFYSPISITSALGMVLLGAKDNTAQQISKILHFDQVTENTTEKAATYHVDRSGN

Pso p27 VHHQFQKLLTEFNKSTDAYELKIANKLFGEKTYQFLQEYLDAIK FYQTSVESTDFANAPEESR

INSWVESQTNEK

SCCA2 81

VHHQFQKLLTEFNKSTDAYELKIANKLFGEKTYQFLQEYLDAIKKFYQTSVESTDFANAPEESRKKINSWVESQTNEKIK

Pso p27

QYNSFNFALLEDVQAKVLEIPYK D

SCCA2 161

NLFPDGTIGNDTTLVLVNAIYFKGQWENKFKKENTKEEKFWPNKNTYKSVQMMRQYNSFNFALLEDVQAKVLEIPYKGKD

Pso p27 LSMIVLLPNEIDGLQK LMEWTSLQNMRETCVDLHLPR

SCCA2 241

LSMIVLLPNEIDGLQKLEEKLTAEKLMEWTSLQNMRETCVDLHLPRFKMEESYDLKDTLRTMGTVNIFNGDADLSGMTWS

Pso p27 AFVEVTEEGVEAAAATAV

SCCA2 321 HGLSVSKVLHKAFVEVTEEGVEAAAATAVVVVELSSPSTNEEFCCNHPFLFFIRQNKTNSILFYGRFSPP

Figure 2b. Peptide sequences detected in the enzyme digests of Pso p27 corresponding to SCCA2.

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2D Gel-electrophoresis and Immunoblot.

Protein staining of Pso p27 after 2-dimensional gel-electrophoresis revealed several

proteins with iso-electric points between 4.7 and 5.3 (Fig 3).

Immunoblot analyses were performed using two murine monoclonal antibodies against

Pso p27 as primary antibodies. The antibodies recognize two different epitopes, and as

demonstrated in Fig 3, both epitopes were recognized in the different variants of the

protein. The modest correlation between the intensity of the silver staining and the

immunoblot is most likely due to variation with respect to exposure of the epitopes.

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Figure 3. 2D- gel-electrophoresis with protein staining and immunoblot using two

different monoclonal antibodies against Pso p27

pH 4.7 pH 5.3

13

Silver

3A3D10

2C7D10

DISCUSSION

Much effort has been put forward in the search for etiological agents associated with

chronic inflammatory - or autoimmune diseases. During the last decades we have

focused on a protein, Pso p27, associated with psoriasis [10-13, 17]. Pso p27 is

expressed in psoriatic lesions and is not detected in uninvolved psoriatic skin or skin

biopsies from healthy controls [10, 13]. Through analysis of antibodies obtained from

psoriatic scale, we have demonstrated the potential role of Pso p27 as an antigen in

psoriasis [10]. The presence of Pso p27 in mast cells in the skin lesions [11] is of

particular interest in view of the potential role of mast cells as immune potentiating cells

[18].

The N-terminal amino acid sequence of Pso p27 described fifteen years ago [19] shows

a clear homology to SCCA [20]. In this paper the relationship between Pso p27 and

SCCA molecules is substantiated. Surprisingly, the Pso p27 antigen seems to represent

various SCCA molecules but with N-terminal and C-terminal ends deviating from the

SCCA molecules. The terminal ends of the Pso p27 antigens are most likely due to a

posttranslational digestion of SCCA molecules with highly specific endoproteases.

The 2-dimensional gel-electrophoresis demonstrates heterogeneity of the Pso p27

antigen with respect to pI. This heterogeneity may be due to variation in amino acid

sequences, but also variation with respect of oxidation of amino acids which was

observed through the mass spectrometry analysis. The two murine monoclonal

antibodies against Pso p27 used in the immunoblot analysis recognize two different

14

epitopes on Pso p27 as they function in sandwich ELISA with Pso p27 as antigen [16].

The fact that all the variants of Pso p27 detected in the 2-dimensional gel-

electrophoresis are recognized by both monoclonal antibodies, demonstrate at least two

common antigenic structures among the variants. The monoclonal antibodies used for

isolation of Pso p27, does not cross-react with the native forms of SCCA 1 or SCCA 2

[21]. Moreover, when the monoclonal antibodies are used in indirect

immunofluorescence analysis of psoriatic skin lesions the antibodies bind primarily to

mast cells and scales, and scarcely to epidermal cells [13] in contrast to anti-SCCA

antibodies [22].

Based on the observations presented in this study it seems reasonable to suggest that a

posttranslational modification of SCCA molecules make them immunogenic and that

the great variability with respect to protein structure is of importance with respect to

failure in immunologic tolerance.

Pso p27 antigen has been shown to participate in the generation of complement

activating immune complexes, both in the psoriatic plaques and in synovial fluid from

patients with psoriasis arthritis [17, 23]. Furthermore, Pso p27 antigen has been detected

in patients with various inflammatory diseases as for example ankylosing spondylitis

[23], sarcoidosis [16] and chronic inflammatory bowel diseases [24].

A highly relevant question is whether the Pso p27 antigen is a common antigenic

principle in chronic inflammatory diseases.

15

ACKNOWLEDGEMENT

The work has been supported by grants from The Research Council of Norway. Thanks

to the FUGE Proteomics Laboratory, Norwegian University of Science and Technology

for assistance with methods and instruments.

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