Báo cáo khoa học: A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin pot

A variety of pro-tein and peptide substrates were processed in vitro; Keywords astacin family; image analysis; Madin–Darby canine kidney cells; meprin; protease proteomics Correspondence

A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

Daniel Ambort1, Daniel Stalder2, Daniel Lottaz1, Maya Huguenin1, Beatrice Oneda1, Manfred Heller2 and Erwin E Sterchi1

1 Institute of Biochemistry and Molecular Medicine, University of Berne, Switzerland

2 Department of Clinical Research, University Hospital, Berne, Switzerland

The astacin-like zinc-dependent

metalloendopepti-dase human meprin (hmeprin) (EC 3.4.24.18) was

first discovered in 1982 for its ability to hydrolyze

N-benzoyl-l-tyrosyl-p-aminobenzoic acid, a

chymo-trypsin substrate used for assessing exocrine pancreas

function [1] N-benzoyl-l-tyrosyl-p-aminobenzoic acid

hydrolase (PPH) was subsequently purified and

charac-terized from human small intestinal mucosa [2] At the

same time, PPH orthologs, called meprin (metal

endo-peptidase from renal tissue) or endoendo-peptidase-2, were

found in mouse and rat kidney, respectively [3,4] Two

similar subunits, termed meprina and meprinb, with

molecular masses of 95 and 105 kDa, respectively,

were identified Human meprin cDNA was expressed

in Madin–Darby canine kidney (MDCK) cells, a

well-established cell system for polarized epithelial cells To date, no such thoroughly characterized model system exists for human epithelial cells Hmeprina is secreted into the culture medium of MDCK cells as inactive homodimers, whereas hmeprinb is primarily mem-brane-bound [5] Hence, heterodimers of hmeprina⁄ b allowed for localization of the a-subunit to the plasma membrane [6] Inactive zymogens of hmeprina and b are processed by limited proteolysis with trypsin into their active forms [5,6] Hmeprina, but not b, may alternatively be activated by plasmin [7,8]

A first step towards the elucidation of the biological function of meprin was achieved by testing putatively cleavable polypeptide substrates A variety of pro-tein and peptide substrates were processed in vitro;

Keywords

astacin family; image analysis; Madin–Darby

canine kidney cells; meprin; protease

proteomics

Correspondence

E E Sterchi, Institute of Biochemistry and

Molecular Medicine, University of Berne,

Bu¨hlstrasse 28, CH-3012 Berne, Switzerland

Fax: +41 31 631 3737

Tel: +41 31 631 4199

E-mail: erwin.sterchi@mci.unibe.ch

(Received 14 April 2008, revised 8 July

2008, accepted 10 July 2008)

doi:10.1111/j.1742-4658.2008.06592.x

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin–Darby canine kidney epithelial cells A simple 2D IEF⁄ SDS ⁄ PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin– Darby canine kidney cells expressing meprin in zymogen or in active form The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS⁄ MS analysis Of 22 (17 nonredundant) candidate substrates identified, the proteolytic process-ing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed

by means of immunoblotting validation experiments The classification of substrates into functional groups may propose new functions for meprins

in the regulation of cell homeostasis and the extracellular environment, and

in innate immunity, respectively

Abbreviations

ADAM, a disintegrin and metalloprotease; BMP-1, bone morphogenetic protein 1; CID, collision-induced dissociation; ECM, extracellular matrix; hmeprin, human meprin (EC 3.4.24.18); ICAT, isotope-coded affinity tag; MDCK, Madin–Darby canine kidney; MMP, matrix

biologically active peptides [2,9], as well as

gastrointes-tinal peptides and extracellular matrix (ECM)

com-ponents, such as collagen type IV, fibronectin and

laminin-nidogen [10–12] These findings suggest that

meprin may be involved in processes such as renal

clear-ance of vasoactive peptides from blood plasma,

regula-tion of cell movement, secretory activity and growth of

intestinal tract, and tissue remodelling In addition,

marked differences between a- and b-subunits in

sub-strate and peptide bond specificity point to distinct

func-tions for the two forms [10] Meprina selects for small

(e.g serine, alanine and threonine) or hydrophobic (e.g

phenylalanine) residues in the P1 and P1¢ sites and

pro-line in the P2¢ position Meprinb prefers acidic amino

acids in the P1 and P1¢ sites and selects against basic

res-idues at P2¢ and P3¢ In conclusion, protease-substrate

discovery executed by these in vitro cleavage assays was

rather haphazard Thus, meprin and its substrate

reper-toire may be studied in a complex biological context to

identify physiologically relevant substrates

The introduction of protease proteomics enabled

identification of protease and protease-substrate

reper-toires on an organism-wide scale by means of proteomic

techniques [13] Using different cell-based systems [14–

16] a variety of hitherto unkown substrates were found

in conditioned media for the metzincin

metalloendopep-tidases, a disintegrin and metalloprotease (ADAM)-17

and matrix metalloproteinase (MMP)-14 Human

plasma was also used to identify substrates for

recombi-nant MMP-14 in a cell-free system [17] Two

methodo-logical platforms were successfully applied for protein

separation: LC-MS⁄ MS and 2D IEF ⁄ SDS ⁄ PAGE

[14–17] These standard techniques were used in

com-bination with lectin-affinity pre-fractionation and

quantitative tags such as isotope-coded affinity tags

(ICAT) or cyanine dyes for differential in-gel

electro-phoresis From these protease proteomic studies, it

became obvious that metalloendopeptidases are key

modulators of diverse signalling pathways and not

merely ECM degrading entities [18] For example, the

major role of the MMP family is the control of cellular

responses critical to homeostatic regulation of the

extra-cellular environment and the immune response [19,20]

We decided to apply protease proteomics to identify

novel physiologic substrates for meprin, aiming to

elucidate its key functions at the cellular level For

the above described techniques, some conceptual

prob-lems may arise: first, ICAT-based approaches compare

pairs of peptides, and therefore it is not possible to

discover cleaved protein fragments with shortened

(non-trypsin-generated) N- or C-termini; second,

nonglycosylated proteins and fragments escape from

lectin-affinity purification We thus designed a simple

2D IEF⁄ SDS ⁄ PAGE-based protease proteomic appro-ach that remedied these limitations and circumvented complicated quantitative and statistical evaluation Hmeprina⁄ b was transfected into MDCK cells and activated in situ by limited trypsin treatment at conflu-ent cell stage Conditioned media of meprin activated and non-activated cells were concentrated with ultrafil-tration and then separated by 2D IEF⁄ SDS ⁄ PAGE A simple 2D IEF⁄ SDS ⁄ PAGE-based image analysis pro-cedure allowed for detection of protein spots unique to 2D gels produced from conditioned media of meprin activated cells LC-MS⁄ MS analysis of candidate substrates confirmed the validity of this protease prote-omic approach for the discovery of shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments

Results

Design and application of a simple 2D IEF/SDS/ PAGE-based protease proteomic approach

in substrate finding Traditionally, 2D IEF⁄ SDS ⁄ PAGE-based image analy-sis is performed on two sets of gels and protein spots are matched to the same reference gel within one single analysis Statistical tools are then applied to quanti-tatively assess subtle but significant changes in peak volumes to find up- or down-regulated protein spots Unfortunately, error-prone matching to wrong refer-ence spots is often underestimated, making quantita-tive statistical information useless Hence, annotations

of interesting candidate spots to wrong spots in the reference gel leads to misinterpretation of the data set and protein spots unique to only one specific condition are then not properly displayed in the corresponding reference gel A remedy to false-positive data interpre-tation is the stepwise reduction in complexity of such

an analysis Therefore, we designed a simple image analysis procedure in which digitized 2D gels were cut into four parts or quadrant sections This procedure enabled the performance of four independent image analyses in which the gel parts of each corresponding quadrant were used to construct four independent ref-erence gels instead of one The corresponding quadrant sections were grouped into sets of gels termed level 1 match-sets for each condition (activated meprin versus non-activated meprin) and then into supersets of level

1 match-sets (higher-level match-sets) (Fig 1) The four level 1 match-sets are the reference gels of the respective quadrants from the 2D gel sections of each condition and the four higher-level match-sets are the reference gels of the two different conditions (activated

meprin versus non-activated meprin) This procedure allowed for subsequent matching of protein spots first

to reference gels of the same condition and thereafter

to reference gels common to both conditions The step-wise annotation of protein spots to two independent levels of reference gels allowed for detection of unique spots in the final higher-level match-sets (Fig 2) These differential spots were unique to one specific condition and absent in the other or vice versa Applying the above procedure to conditioned media of MDCKa⁄ b cells revealed that, among 817 protein spots displayed,

35 were unique to media of cells expressing activated meprina⁄ b and 40 to media of cells with non-activated meprina⁄ b (Table 1) These unique protein spots were therefore absent in the corresponding other condition Thus, unique spots were indicative of proteins released into or proteolytically cleaved in the extracellular milieu by hmeprina⁄ b We then hypothesized that, upon LC-MS⁄ MS analysis of candidate substrates, it may be feasible to find shortened (non-trypsin-gener-ated) N- and C-termini in peptide fragments Such potential N- or C-terminally truncated cleavage prod-ucts can be identified in protein spots unique to condi-tioned media of trypsin activated MDCKa⁄ b cells, as shown below

The procedure is based on qualitative differences among reference

gels (level 1 match-sets) of each group of five gel replicates (three

pooled biological gel replicates and two more technical gel

repli-cates) Gel replicates of each group (activated meprin versus

non-activated meprin) were cut virtually into four equally spaced

quadrants for four independent image analyses Reference gels of

each group were then clustered into a new set for higher-level image

allowed for detection of unique protein spots The combined

higher-level match-set is the final fusion of all annotated unique spots into

one big 2D reference map.

of the first quadrant is shown Two hundred and fifty micrograms of conditioned medium protein from trypsin activated and non-activated

Optimized Ruthenium staining: for each condition (activated meprin versus non-activated meprin), three pooled biological gel replicates (from

18 dishes per pooled sample) and two more technical gel replicates (of one pooled sample) were produced for subsequent image analysis Unique protein spots are labelled in level 1 and higher-level match-sets with SSP assigned by the image analysis software.

Protein identification by means of LC-MS/MS,

PHENYX-based andBLASTP-based protein database

searching

By visual inspection, the 35 protein spots unique to

media of trypsin activated MDCKa⁄ b cells could be

reduced to 33 putative candidates The redundancy of

two spots present in more than one quadrant from

each set of 2D gels analysed prompted correction

(Fig 2; see Fig S1) On colloidal Coomassie stained

preparative 2D gels, 24 protein spots of interest were

detectable These spots could be rematched to putative

candidates found in fluorescence stained analytical

gels (data not shown) Gel plugs were then prepared,

in-gel digested with trypsin and peptides thereof

separated⁄ fragmented by LC-MS⁄ MS

Collision-induced dissociation (CID) spectra interpretation with

phenyx (version 2.1) against the uniprot-SwissProt

protein database (release 48.8) led to 22 (17

nonredun-dant) protein identifications (Fig 3 and Table 2) The

taxonomic search space was restricted to Mammalia

(40 084 sequence entries) To double-check significant

hits, the same spectra were interpreted with the

web-based search engine mascot (version 2.1) against the

same database and parameter settings (data not

shown) [21] The identification of nucleophosmin

(pro-tein spot SSP 2102; Table 2) was accepted because the

peptide VDNDENEHQLSR and its in-source

pro-duced fragment DNDENEHQLSLR were

unambigu-ously identified with good scores by phenyx and

mascot In addition, the whole tryptic peptide MSVQPTVSLGGFEITPPVVLR was identified by phenyx and mascot as first ranking identification, but with scores below the chosen acceptance criteria (Table 2 and data not shown) Beside six positive hits for dog, other species (e.g rat, human, rabbit and mouse) were predominantly represented The current release (51.3) of the uniprot-SwissProt protein data-base lists 664 sequence entries for dog and thus may explain the poor representation in this species Recently, the dog genome was sequenced to comple-tion [22] Peptide sequence tags deciphered from our previous analysis permitted search with blastp (version 2.2.16) against the 33 527 dog RefSeq protein sequence entries of the NCBI [21] All top scoring significant hits corresponded to predicted dog protein sequence entries Finally, all equivocal uniprot-SwissProt protein database searches were successfully matched to pre-dicted dog protein orthologs (Table 3)

Discovery of shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products

in peptide fragments phenyx offers the remarkable feature to search for non-tryptic peptides (i.e half-cleaved peptides) In-gel tryptic digestion of proteins contained within gel plugs produces peptide fragments terminating C-terminally with a lysine or arginine residue Trypsin cleavage specificity is then fixed to the N- or C-terminus

Qualitative spot matching differences among reference gels (level 1 match-sets) are expressed as unique spots (% of each corresponding quadrant section).

Unique spots (%)

Level 1 match-set

Higher-level match-set

In silico digestion of the theoretical full-length protein

product with trypsin enables the determination of all

tryptic peptides terminating with a lysine or arginine

residue Hence, peptide fragments not featuring a lysine or arginine residue in the C-terminal ends or truncated in the N-termini by some amino acids rela-tive to the preceding in silico-generated tryptic frag-ments are candidates for proteolytically processed (non-trypsin-derived) cleavage products In a protease proteomic approach, this option facilitates the discov-ery of shortened (non-trypsin-generated) N- or C-ter-minally truncated cleavage products defined by meprin protease activity To determine new peptide ends other than lysine or arginine, peptides must not be identified either C- or N-terminal to the truncated peptide We applied this strategy to all protein database searches performed with phenyx Several shortened half-cleaved peptides (not full-length tryptic peptides) were detected (Table 2) Half-cleaved peptides may also originate from in-source fragmentation of intact tryptic peptides during the ionization process Accordingly, the follow-ing half-cleaved peptides co-eluted with correspondfollow-ing intact tryptic peptides after chromatographic separa-tion: TDGNSEHLKR and DGNSEHLKR from pro-tein spots SSP 602⁄ 9602 and SSP 1602, respectively; PGPVFGSK from protein spot SSP 1602; and DNDE-NEHQLSLR from protein spot SSP 2102 The half-cleaved peptides derived from the sequence stretching over amino acids 159–182 of clusterin (IDSLLENDR-QQTHALDVMWDSFNR) found in protein spots SSP

502 and SSP 1502 were chromatographically separated and thus may not refer to in-source fragmentation products Those half-cleaved products are most proba-bly related to in-gel digestion artefacts because cleav-age within this protein sequence stretch by meprin must be excluded due to an overall amino acid sequence coverage of this protein that exceeded amino acid 182 In addition, the two half-cleaved peptides DQAVSDTELQEMSTEGSK (residues 23–40) and DTELQEMSTEGSK (residues 28–40) in SSP 502 and

1502 were chromatographically separated and were not in-source fragmentation products generated during the ionization process The former peptide represented the mature N-terminus of clusterin (aspartate at position 23) and hence was not generated by meprin activity The latter peptide was presumably produced by meprinb with acidic amino acids preferred in the P1¢ position and selecting against basic amino acids in the P2¢ and P3¢ positions [10] The leguminous lectin-like VIP36 was present in two different protein spots (SSP

1602 and SSP 602⁄ 9602) and also met our criteria for shortened (non-trypsin-generated) C-terminally trun-cated cleavage products in peptide fragments In both spots, the truncated peptide LFQLMVEH (residues 273–280) was identified with no further peptides towards the C-terminal end (not ending with a lysine

A

B

C

Fig 3 Two-dimensional reference maps on protein identifications.

Representative 2D gel images of conditioned medium protein from

of non-activated meprin Unique protein spots were labelled with

SSP defined by image analysis software (C) Close-up view of one

analy-sis of candidate substrates confirmed the validity of this protease

proteomic approach for the discovery of shortened

(non-trypsin-generated) N- and C-terminally truncated cleavage products in

peptide fragments (Table 2).

Protein identification

SwissProt accession number

Number of

Experimental m

Theoretical mass

Peptide z-score

Peptide P

Protein identification

SwissProt accession number

Number of

Experimental m

Theoretical mass

Match delta m

Peptide z-score

Peptide P

Protein identification

SwissProt accession number

Number of

Experimental m

Theoretical mass

Match delta m

Peptide z-score

Peptide P

or arginine residue) Additionally, this truncated pep-tide was not generated by in-source fragmentation because there was no co-eluting ion trace of the corre-sponding whole tryptic peptide LFQLMVEHTPDEE-NIDWTK VIP36 was described as a single-pass type I membrane protein with an extracellular carbohydrate recognition domain exactly terminating at those amino acids (residues 52–280) [23] Moreover, the amino acid sequence following the putative cleavage site corre-sponded to cleavage preference for meprina with the amino acids threonine and proline in the P1¢ and P2¢ positions [10] The targeted cleavage by hmeprin after this specific domain may indicate protein ectodomain shedding Nevertheless, the biological consequence of this remains to be elucidated

Functional clustering into biological process and molecular function

Next, the proteins identified by LC-MS⁄ MS as puta-tive meprin substrates were classified into functional groups according to the Human Protein Reference Database (Table 3) [24] Ten proteins could be assigned to the biological process of ‘cell growth and⁄ or maintenance’ and four to ‘immune response’ (Fig 4) The remaining proteins were equally distrib-uted into functional classes such as ‘transport’, ‘cell communication; signal transduction’, ‘metabolism; energy pathways’ and ‘protein metabolism’ In conclu-sion, these findings suggest possible functions for meprin in the regulation of cell homeostasis and the extracellular environment, and in the immune response

Effect of in situ trypsin treatment Zymogen activation by limited trypsin treatment may lead to changes elicited by the trypsin and not by meprin To exclude such unspecific side effects caused by the trypsin treatment rather than by the effector (membrane-bound hmeprina⁄ b), wild-type (WT) and meprina⁄ b MDCK cells were treated in the same way Media of trypsin-treated and non-treated cells were prepared and then subjected to 1D SDS⁄ PAGE and subsequent densitometric image analysis with aida software (Fig 5) We decided to perform a comparison between conditioned media of

WT and meprina⁄ b MDCK cells on 1D gels Quan-titative assessment of protein bands revealed no significant differences between trypsin-treated and nontreated WT samples, whereas meprina⁄ b samples showed substantial differences upon trypsin activa-tion Moreover, the protein patterns of WT versus

Protein identification

SwissProt accession number

Number of

Experimental m

Theoretical mass

Match delta m

Peptide z-score

Peptide P

a SS

f Ma

i No

j

k Sh

l Ha

meprina⁄ b differed as well (Fig 5A) and indicated

that overexpression of hmeprina⁄ b per se causes

dif-ferences that are independent from zymogen

activa-tion Finally, triplicate image analysis of gel lanes

confirmed these findings (Fig 5B) but, more

impor-tantly, revealed a trend towards the appearance

of low molecular weight proteins in media of

meprina⁄ b MDCK cells Hence, the triplicate

assess-ment of data generated unambiguously pointed to

reproducible differences triggered by the activation

and not by the overexpression of meprina⁄ b (Fig 5C) Obviously, activation of meprina⁄ b results

in the release of proteins into the culture medium

Validation of direct or indirect effects by immunoblotting follow-up experiments Proteomics is a very powerful tool for protease-substrate identification, but the data obtained need to

be verified by means of alternative techniques Western

at NCBI Functional classification according to Human Protein Reference Database.

NCBI accession

Expected

PREDICTED: similar to

annexin A1

Cell communication;

signal transduction

PREDICTED: similar to

collagen alpha 2(V)

chain precursor

maintenance

Extracellular matrix, structural constituent

PREDICTED: similar to

elongation factor 2

regulator activity

PREDICTED: similar to

filamin A isoform 8

maintenance

Cytoskeletal anchoring activity

PREDICTED: similar to

fructose-bisphosphate

aldolase A isoform 2

Metabolism; energy pathways

PREDICTED: similar to

maintenance

Structural molecule activity

PREDICTED: similar to

macrophage capping protein

maintenance

Cytoskeletal protein binding

PREDICTED: similar to

nucleophosmin 1 isoform 12

PREDICTED: similar to

phosphoserine

aminotransferase isoform 1

Metabolism; energy pathways

PREDICTED: similar to

protein-lysine 6-oxidase

precursor isoform 3

maintenance

PREDICTED: similar to

stanniocalcin-1 precursor

Cell communication;

signal transduction

PREDICTED: similar to

thrombospondin 1 precursor

maintenance

Extracellular matrix, structural constituent

maintenance

Cytoskeletal protein binding

a

nearly exact matches’: word size 2, filter off, expect value 20 000, score matrix PAM30.