Carbohydrate Research 389 (2014) 72–77

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Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Bacterial expression and functional reconstitution of humanheparanase

http://dx.doi.org/10.1016/j.carres.2014.01.0020008-6215/� 2014 Published by Elsevier Ltd.

Abbreviations: ECM, extracellular matrix; GAG, glycosaminoglycan; HSPG,heparan sulfate proteoglycan; Hepsmaf, small subunit of human heparanase;Heplaf, large subunit of human heparanase.⇑ Corresponding author. Tel.: +43 316 380 5373; fax: +43 316 380 9846.

E-mail address: andreas.kungl@uni-graz.at (A.J. Kungl).

Sophie Winkler a, Daniela Schweiger a, Zheng Wei b, Erich Rajkovic a, Andreas J. Kungl a,⇑a Department of Pharmaceutical Sciences, University of Graz, Humboldtstrasse 46, 8010 Graz, Austriab Department of Medical Oncology, Paterson Institute for Cancer Research, Manchester M204BX, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 September 2013Received in revised form 3 January 2014Accepted 4 January 2014Available online 14 January 2014

Keywords:Human heparanaseAngiogenesisTumor metastasisExtracellular matrixHeparan sulfate

Human heparanase is a heparan sulfate degrading enzyme located in the extracellular matrix playing adecisive role in angiogenesis and tumor metastasis. Translated as a 65 kDa inactive prae-form, the proteinis processed into an 8 kDa and a 50 kDa subunit which form a non-covalently associated active hetero-dimer. We have expressed the two subunits separately in Escherichia coli which yielded active humanheparanase upon reconstitution. The two purified subunits folded independently and secondary structureanalysis by far-UV CD spectroscopy gave 33.1/11.1% a/b content for the 50 kDa subunit and 6.9/49% a/bcontent for the 8 kDa subunit. This heparanase expression system is easy and can be used for efficientscreening for enzyme inhibitors.

� 2014 Published by Elsevier Ltd.

1. Introduction

Angiogenesis and tumor metastasis take place in the structur-ally heterogeneous and complex extracellular matrix (ECM) envi-ronment and are therefore strongly influenced by the ECMorganization and composition. Remodeling of the extracellular ma-trix in terms of modulating endothelial and vascular cell behavioris a major prerequisite for the formation of new blood vessels.1

This necessitates the initial breakdown of the components of theextracellular matrix and the sub-endothelial basement membrane,the amorphous and dense division to the blood vessel.2

Proteoglycans are major components of the ECM and they arefound on almost all mammalian cells and tissues. They are com-posed of glycosaminoglycan (GAG) chains covalently attached toa core protein by a serine linker.3–6 While the protein part isresponsible for the localization of the proteoglycan, for examplein the cell membrane, in the ECM or in the specialized basementmembrane, the GAG constituent mediates the functional interac-tions. The backbone of the glycosaminoglycan-chains consists ofdisaccharide repeating units of glucosamine linked to either aniduronic or a glucuronic acid7 and the functional complexity is

ensured by the introduction of diverse modification like sulfationor acetylation at the 2-O, 3-O, 6-O or the N-position. Due to thesevariations along the entire polysaccharide chain, the heparan sul-fate proteoglycans interact with a great variety of ligands, amongthem is the large family of growth factors which is decisive fortumor progression and tumor angiogenesis.8–10

In order to maintain the delicate balance between angiostaticand angiogenic stimuli and to respond rapidly to changes in theECM environment, these bioactive molecules are stored in aninactive form by binding to diverse species of heparan sulfate pro-teoglycans (HSPGs).10 It has been found that invading cells, espe-cially metastatic and tumor cells, but also leukocytes have theability to traverse ECM barriers and basement membranes bysecreting large amounts of degrading enzymes like matrix metallo-proteinases, serine-, cysteine-, and aspartic-proteases and in par-ticular, the heparanase enzyme.11,12

So far, several groups reported independently the cloning of onesingle human heparanase cDNA encoding a 65 kDa latent pre-proheparanase.13–16 This inactive form of the enzyme is processedresulting in the loss of the signal peptide and the excision of a6 kDa intervening peptide which finally allows the formation ofthe active heterodimer consisting of the 8 kDa and the 50 kDa sub-unit.17 This active heparanase enzyme has been detected in severalmalignant cells and tissues and is responsible for tumor angiogen-esis and metastasis.18,19 Due to this reason the enzyme was identi-fied to be a promising target for therapeutically interfering with

S. Winkler et al. / Carbohydrate Research 389 (2014) 72–77 73

cancer diseases20 and diverse efforts have been made to inhibit itsactivity by the generation of novel heparan sulfate mimetics.12

Therefore finding a specific molecular inhibitor of the heparanasestill remains one of the major aims in the field which may bereachable by screening natural compound libraries or by designinga competitive structure towards the natural HS substrate of theenzyme. For this purpose, structural and biophysical data of theenzyme are highly desired.

In this report we present the cloning, expression, purification,and functional reconstitution of human heparanase in Escherichiacoli as well as secondary structural data. Our bacterial expressionsystem can be used to efficiently screen for enzyme inhibitorswithout requiring downstream processing of the enzyme subunits.

Figure 1. SDS–PAGE of the purified 8 kDa and 50 kDa subunit. (A) 15% SDS–PAGEgel of the 8 kDa subunit followed by silver staining. (B) 10% SDS–PAGE gel of the50 kDa subunit followed by silver staining.

2. Materials and methods

2.1. Materials

All used materials were purchased from Sigma-Aldrich (St.Louis, MO, USA) unless otherwise specified. The vector p2134 con-taining the full length cDNA of the Human Heparanase encoding543 amino acids was a kind gift of SANOFI-Synthelabo Laboratoire(Toulouse, France).

2.2. Cloning of 8 kDa fragment (Hepsmaf) and 50 kDa fragment(Heplaf) into bacterial expression plasmid

The 8 kDa subunit was generated from amino acid Gln36 to Glu108

by PCR amplification by using TaqBiotherm polymerase (genXpress,Austria), vector p2134 as DNA template containing the full lengthhuman heparanase cDNA and the primers 8Hepfwd 50-CAGGACGTCGTGGACCT-30 and 8Heprev 50-TTCCTTCTTGGGATCGAAAA-30.The 50 kDa subunit including the amino acid sequence from Lys158

to Lys514 was amplified by PCR using TaqBiotherm (genXpress),vector p2134 as DNA template and the primer 50HepFWD 50-AAAAAGTTCAAGAACAGCACCTAC-30 and 50HepREV 50-TTTTTCCATTAAAGGTGGCAAG-30. Both amplified products were ligated intopCR�T7/NT-TOPO�-vectors (Invitrogen Corporation, Carlsbad,California, USA) for expression in E. coli as an N-terminal 6xhisti-dine-tagged fusion protein. The pCR�T7/NT-TOPO� heparanase sub-unit plasmids were transformed into TOP10 E. coli strain (purchasedfrom Invitrogen). Prior to expression the correct orientations andsequences of the constructs were verified by DNA sequencing.

2.3. Expression of the Hepsmaf and Heplaf

Plasmid constructs were transformed into the E. coli expressionstrain BL21 (DE3)plysS (Invitrogen). 16 L LB cultures, each inocu-lated with a single colony, were grown under shaking (200 rpm)at 37 �C containing 100 lg/mL ampicillin to an optical cell density(A600) of 1.2. Protein expression was induced by addition of 1.0 mMisopropyl-b-D-thiogalactopyranoside (IPTG) (genXpress, Austria).The cell cultures were kept for shaking for another 3 h at 37 �C tocomplete protein expression. 1 mL probes were taken before andin one hour intervals after induction with IPTG to analyse overex-pression of the subunits by SDS–PAGE and coomassie-blue stain-ing. The cells were harvested by centrifugation at 6000�g for15 min at 4 �C. The pellet was then resuspended in lysis buffer con-taining 8 M urea, 50 mM Na2HPO4 pH 7.5, and 300 mM NaCl. Sub-sequently, bacterial cells were lysed by moderate ultrasonication(3 times for 10 s) on ice and centrifuged for 45 min at 20,000�gto fractionate the soluble proteins from the insoluble elements.The clear supernatant was analyzed by SDS–PAGE and coomas-sie-blue staining for the presence of either the 8 kDa or the50 kDa subunit.

2.4. Purification of the 8 kDa subunit (Hepsmaf)

The smaller fragment of the human heparanase was purified tohomogeneity in one step followed by gradient/stepwise dialysis.Therefore the crude denatured lysate was applied to a 4 mLTALON� metal affinity resin (CLONTECH Laboratories, Palo Alto,CA) column with a flow rate of 0.3 mL/min after system equilibra-tion with wash-buffer (8 M urea, 50 mM Na2HPO4 pH 7.5 and100 mM NaCl). Unspecific bound material was first removed byextensive washing with wash-buffer followed by a second washstep with low imidazole concentrations (25 mM) to further reduceprotein contaminations due to unspecific interactions. The tightlybound his-tagged fusion protein (8 kDa fragment) was then elutedwith elution-buffer containing 8 M urea, 50 mM Na2HPO4 pH 7.5,100 mM NaCl, and 150 mM imidazole. The collected and pooledfractions were dialyzed in a 5 step gradient from 8 M urea to PBSpH 7.4 for further analysis. With this protocol a purification of95% was achieved, confirmed by SDS–PAGE and silver staining21

(see Fig. 1). Protein identification was performed by Westernblotting using an anti-HisG-antibody (Invitrogen Corporation,Carlsbad, California, USA) and MS/MS analysis. Protein concentra-tion was determined by the BCA22 Protein Assay Reagent Kit(Pierce Biotechnology, Rockford, IL, USA) according to the manu-facturers instructions.

2.5. Purification of the 50 kDa subunit (Heplaf)

The crude denatured protein lysate was loaded onto a TALONcolumn (as described above). The 50 kDa fragment containing frac-tions were pooled and diluted to a final solution of 8 M urea,50 mM Na2HPO4 pH 10.60, and 25 mM NaCl (150 mM imidazolewere removed by dialyzing). This lysate was loaded on aQFF-Sepharose (3 mL) column (Amersham Biosciences, Uppsala,Sweden) with a flow rate of 0.5 mL/min. After reaching the baselineby washing with wash-buffer (8 M urea, 50 mM Na2HPO4 pH10.60, and 25 mM NaCl) a gradient was started towards native buf-fer conditions (50 mM Na2HPO4 pH 10.60, and 25 mM NaCl). The50 kDa subunit was eluted from the ion-exchange column by a sec-ond gradient towards high salt concentrations (elution-buffer:50 mM Na2HPO4 pH 10.60 and 1.3 M NaCl). The protein was elutedat about 65% elution buffer (corresponding to 845 mM NaCl) over 2

74 S. Winkler et al. / Carbohydrate Research 389 (2014) 72–77

fractions. The purity of the protein was determined by SDS–PAGEand silver staining (>95% pure; see Fig. 1). Protein identificationwas performed by MS/MS-based trypsin-derived peptide profilingand by western blotting with anti-HisG antibody (data notshown).23–25 Protein concentrations were determined as describedabove.

2.6. Labeling of heparan sulfate for activity assay

Heparan sulfate (sodium salt) from bovine kidney was labeledwith fluorescein isothiocyanate (FITC). 5 mg of heparan sulfateand 5 mg of FITC were dissolved in 1 mL of 0.1 M sodium carbonatebuffer pH 9.5 and stirred at 4 �C in darkness over night. The solu-tion was then loaded on a PD-10 desalting column to isolateFITC-labeled heparan sulfate (FITC-HS) from free FITC. The FITC-HS was further subjected to chromatography through SephacrylS-300 HR equilibrated with 25 mM Tris–HCl, 150 mM NaCl pH7.5 to fractionate high molecular weight products (Mr > 30,000).The fractionated materials were concentrated with a Microcon�

10 concentrator (Amicon) and the quantity of FITC-HS was mea-sured using the Blyscan proteoglycan and glycosaminoglycan assay(Biocolor Ltd, Belfast, Ireland).

5 10

LU

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FLD1 A, Ex=494, Em=520

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Figure 2. Activity assay of the two reconstituted subunits using crude bacterial lysates. (and C stand for the fluorescent materials from the bacterial lysates; the control profilesulfate. Comparing the 0 h and 24 h of digestion, the peak of intact FITC-HS (A) decreas

2.7. Heparanase activity assay

The activity of the enzyme was proved by degradation of FITClabeled heparan sulfate as reported previously.16 The reactionwas carried out in 100 lL of 50 mM sodium acetate pH 4.2, con-taining 0.5–1 lg FITC labeled heparan sulfate. The protein materialcontaining recombinant heparanase subunits (bacterial lysates)was added to the reaction mixture to a total volume of 200 lLwithout exceeding the salt concentration of 0.25 M and incubatedat 37 �C for an appropriate period (in general 24 h). The reactionwas then stopped by the addition of 100 lg of heparin and subse-quent heating at 100 �C for 5 min. The reaction mixture was centri-fuged at 15,000�g for 5 min to precipitate the insoluble material.The products of FITC-HS yielded by this reaction were analyzedby high speed gel permeation chromatography and detected by afluorescence spectrophotometer (see Fig. 2). Briefly, a 20 lL aliquotof the supernatant was injected into a TSK gel G3000SWXL column(7.8 mm inner diameter � 30 cm) equilibrated with wash-buffer(25 mM Tris–HCl, 150 mM NaCl pH 7.5) and run at 0.5 mL/min.The fluorescence in the eluent was measured by an F-1050 fluores-cence spectrophotometer (Hitachi, Tokyo, Japan). The activity wasdetermined from HPLC chromatograms by measuring a forward

mi15 20

2515 20

0 hr

24 hr

B1 C

B C

0 h) Peak A represents the fluorescence of intact FITC labeled heparan sulfate. Peak Bis not shown here. (24 h) Peak B1 contains the degraded amount of FITC heparan

es and the peak containing degraded heparan sulfate (B1) increases.

S. Winkler et al. / Carbohydrate Research 389 (2014) 72–77 75

half area of the peak of the intact FITC-HS. The decrease of this areafollowing heparanase treatment was measured by using an inte-grator, and the amount of the degraded FITC-HS was calculatedfrom the decrease of the fluorescence intensity (FI). All activitytests were performed with crude bacterial lysates containing theoverexpressed subunits.

2.8. Circular dichroism spectroscopy and analysis

All Circular dichroism (CD) spectra were recorded on a JascoJ-710 spectropolarimeter over a range of 195–250 nm in PBS usinga quartz cuvette with a layer thickness of 1 mm. The far UV CDmeasurements were carried out with a response time of 1 s anda data point resolution of 0.2 nm. Three scans were averaged inorder to obtain smoothed data. All spectra were backgroundcorrected and calculated from millidegrees to units of mean

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Figure 3. CD spectra of the 50 kDa subunit of human heparanase. CD spectra of a 707 nMellipticites of the spectra averaged over three scans. The inset (right above) shows a raw dperformed by predict protein28 is shown: E refers to extended strands (beta sheets), and

residue ellipticity using JASCO standard analysis. Data analysisaccording to secondary structure contents was performed usingthe algorithm SELCON.26,27 The Heplaf was analyzed in a 707 nM,the Hepsmaf fragment in a 2.75 lM solution. Different glycosamino-glycans, namely fractioned heparin (average molecular weight5000 Da) and heparan sulfate (average molecular weight about12,000 Da; obtained by Fluka (Buchs, Switzerland)), were addedin a 5 to 10-fold molar excess to the protein solutions. Graphicalrepresentations were performed in ORIGIN-software (MicrocalInc.).

3. Results and discussion

Our goal was to establish an expression system with whichhuman heparanase can be expressed in high yields, at low costwhich can be easily adapted for enzyme inhibitor screening. Since

SGLDLIFGLNALLRTADLQWNSSN

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solution of the large subunit (50 kDa subunit) at 25 �C. Shown are the mean residueata spectrum. Below the sequence of the protein, the secondary structure prediction

H refers to alpha helices.

76 S. Winkler et al. / Carbohydrate Research 389 (2014) 72–77

bacterial expression systems are still amongst the cheapest andmost efficient heterologous production systems for recombinantproteins and since the full-length human heparanase was not suit-able for such an approach (no proteolytic processing of the precur-sor protein in bacteria), we aimed to express the two subunits ofhuman heparanase independently in E. coli. Applying a typicalHis-tag guided affinity purification procedure for both subunits,our approach proved to yield pure proteins (purity >95%, seeFig. 1) with yields which are promising for scale up if larger quan-tities of the enzyme are needed (estimated overall yields: 20% forthe small, and 15% for the large subunit).

In a next step we wanted to investigate whether the two sub-units give a biologically active enzyme after reconstitution. For thispurpose, we first mixed the two bacterial lysates expressing thesmall and large heparanase subunit. We then applied a heparan su-fate degradation assay separating FITC-labeled GAGs according totheir molecular weight on a size-exclusion column. As can be seenin Figure 2, an additional (or larger) peak (B1 in Fig. 2) occurs after24 h in the chromatogram which contains the degraded heparansulfate. Combining the purified subunits resulted in a significantlydecreased enzymatic activity (data not shown). We interpret theseresults by the lack of post-translational modifications (mainly gly-cosylations) on recombinant proteins expressed in E. coli. Thesemodifications are usually important for conformationally stabiliz-ing proteins, thereby improving their stability and decreasing theirtendency towards aggregation, both of which are important forbiological function. In crude bacterial lysates, bad solubility andaggregation are prevented by other bacterial proteins which there-fore gave a very good heparanase activity in the mixed lysates,whereas reconstituting the purified two subunits gave less goodactivity.

QDVVDLDFFTQEPLHLVSPSFLSEEEEE EE

ARGLSPAYLRFGGTKTDFLIFDPEEEEHH EEEE

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Figure 4. CD spectra from the 8 kDa subunit of human heparanase. Smoothed CD spectabove; the inset shows the original spectrum. Below the sequence of the protein, the sextended strands (beta sheets), and H refers to alpha helices.

Next we investigated the secondary structure of the two hepa-ranase subunits by far-UV circular dichroism (CD). The spectra ofthe 8 kDa and the 50 kDa domain which were subsequently ana-lyzed using the SELCON algorithm are shown in Figures 3 and 4(details see Section 2). In addition, theoretical predictions of thesecondary structure were performed using the internet service ofPredictProtein.28 In the case of the large fragment, a protein con-centration of 707 nM was sufficient to obtain an adequate CD sig-nal, the small subunit needed higher protein concentration ofabout 2.75 lM due to the lower number of peptide bonds. SELCONanalysis was performed for the background corrected spectra giv-ing a secondary structure content closely related to an (alpha/beta)8 TIM-barrel fold: 33.1% and 11.1% of the residues of the Heplaf

exhibit alpha-helix and beta-sheet, respectively, and 54.7% unde-fined coils (see Fig. 3). These results correspond well with the dataof secondary predictions, namely 28.8% helix, 23.8% beta-sheet,and 47.4% others. The 8 kDa domain Hepsmaf was found to foldindependently of the large subunit. SELCON analysis of the smalldomain revealed an almost all beta fold with only 6.9% of the ami-no acids residing in alpha helices and 49% in beta sheets (58.1%were found in other elements, see Fig. 4).

Because of their enormous complexity, heparan sulfates areable to interact with a broad spectrum of bioactive molecules suchas growth factors. These proteins are tethered to the glycosamino-glycan part of proteoglycans and are by this way inactivated.Through the degradation of heparan sulfate, growth factors andother signaling factors become released and start their interplaywithin the extracellular matrix. With regard to angiogenesis, thehuman heparanase plays a crucial role in the initiating steps ofnew blood vessel formation from already existing vessels. Sinceinvading tumor cells can recruit and also produce the heparanase

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S. Winkler et al. / Carbohydrate Research 389 (2014) 72–77 77

enzyme to ensure supply of oxygen and nutrients during solid tu-mor growth, inhibition of this enzyme seems to be a promisingnew way of cancer therapy. For the purpose of drug screening, de-sign and development, however, reasonable amounts of the hepa-ranase target protein need to be available. We have presented herea novel method for the delivery of recombinant biologically activehuman heparanase and we have shown that the two subunits ofthe enzyme are obtained at high yields and are properly foldedafter downstream processing. Moreover, reconstituting the twobacterial lysates expressing the two heparanase subunits resultedin good enzyme activity which can be used to efficiently screenfor heparanase inhibitors.

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