A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding

Abstract

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.

Figure 1. Analysis of FSAP activity in the presence of total RNA, genomic DNA, tRNA and heparin

(A) The activity of FSAP (20 nM) was analysed in the presence of different concentrations of total RNA (▲), genomic DNA (△), tRNA (●) and heparin (■). FSAP activity is expressed as substrate S-2288 turnover (μM/min) at maximal reaction velocity. (B) Biotinylated heparin (2 μg/ml) was mixed with dilutions of total RNA (▲), tRNA (●) and heparin (■), and these mixtures were added to FSAP-coated wells for 1 h. The wells were washed and biotinylated heparin binding was measured. Results represent means±S.D.

Figure 2. SDS/PAGE analysis of human FSAP hydrolysed by in situ generated plasmin

(A) Purified human FSAP (lane A) consists of approx. 98% inactive single-chain (sc) and 2% active two-chain form composed of the heavy chain (hc, amino acids 23–313) and the light chain (lc, amino acids 314–560). Upon incubation in PBS (pH 7.4), human FSAP undergoes complete autoactivation followed by further inactivation by hydrolysis of the light chain, resulting in fragments of approx. 16 and 10 kDa (lane B). Incubation of human FSAP with plasminogen and uPA (leading to in situ generation of plasmin) resulted in a complex hydrolysis pattern (lane C). Without incubation of the samples with ME, several chains of hydrolysed FSAP are connected through disulphide bridges. (B) For N-terminal sequencing, hydrolysed human FSAP was incubated in the presence (lane 1, 50 μg of protein) or absence (lane 2, 5 μg of protein; lane 3, 50 μg of protein) of ME for 5 min at 95 °C respectively and separated by SDS/PAGE. The protein fragments were blotted on to a PVDF membrane and stained with Coomassie Blue. Bands F1–F19 were analysed by N-terminal sequencing. Molecular mass standards are indicated in kDa.

Figure 3. Interaction of hydrolysed human FSAP with radiolabelled total RNA

Hydrolysed human FSAP was incubated in the absence (lane A) or in the presence (lane B) of ME for 5 min at 95 °C, separated by SDS/PAGE and blotted on to a PVDF membrane. Proteins were stained with Coomassie Blue, photographed, completely destained with 70% (v/v) ethanol, and equilibrated in binding buffer. The membrane was incubated overnight at 25 °C in the presence of γ-³²P-labelled total RNA. After extensive washing, radiolabelled RNA bound to the membrane was detected by autoradiography. Bands interacting with RNA are indicated with an arrow. Band F17 was unable to bind RNA and is indicated with an asterisk. Bands F10, F11, F12, F13, F14, F15 and F16 showed a high binding capacity for RNA and band F7 showed a weak binding capacity for RNA. Intact disulphide bridges were necessary for efficient RNA binding. Molecular mass standards are indicated in kDa.

Figure 4. Heparin-binding analysis of hydrolysed human FSAP

Hydrolysed human FSAP (hydr. FSAP, −ME) eluted from the heparin column at a similar ionic strength as native human FSAP (native FSAP). In contrast, there was no heparin binding of hydrolysed FSAP when pretreated with 100 mM ME for 15 min at room temperature (hydr. FSAP, +ME). Elution was followed by analysis of UV absorbance at 280 nm. Conductivity indicates the increase in NaCl concentration. SDS/PAGE analysis of the flow-through (lane 1), wash fraction (lane 2) and elution peak (lane 3) was performed and the proteins were detected by silver staining. F11 and F12 bands interacting with heparin are indicated with an arrow and molecular mass standards are indicated in kDa.

Figure 5. Interaction of hydrolysed human FSAP with radiolabelled RNA in solution

[α-³²P]UTP-labelled poliovirus RNA was incubated at 30 °C for 10 min in the absence (lane 1) or presence of hydrolysed human FSAP (lane 2) in solution. Samples were irradiated with UV light (254 nm), treated with 0.1 mg/ml RNase A, denatured and separated on a denaturing SDS/12% polyacrylamide gel. For detection of protein bound to radiolabelled RNA, an X-ray film was exposed for 16 h. Size markers indicate positions of ¹⁴C-labelled marker proteins (M). Arrows indicate the bands F1 and F2. Molecular mass standards are indicated in kDa.

Figure 6. Model of the three-dimensional structure of the EGF3 domain of human FSAP

The available solution structure of the closely related EGF domain of human tPA (PDB code 1tpg) was used to model EGF3 of FSAP using SWISS-MODEL. Residues Arg¹⁶⁷ to Lys¹⁷³, Lys¹⁸³ and Lys¹⁸⁵ are indicated.

Figure 7. Schematic structure of human FSAP and the polyanion-binding domains

(A) The structure of FSAP including identified proteolysis sites (indicated by vertical arrows) and selected forms of FSAP capable of RNA binding are shown schematically. Experimentally identified N-terminal ends are indicated by horizontal arrows and the predicted C-terminal ends are indicated by open circles. The amino acid stretch from residue Arg¹⁶⁷ to residue Lys¹⁷³ (RHKRRSK) has a high average positive charge and is also the site within the EGF3 (E3) domain that is essential for RNA binding, since deletion of the amino acids Arg¹⁷⁰, Arg¹⁷¹, Ser¹⁷² and Lys¹⁷³ in hydrolysed FSAP (F17) resulted in complete loss of RNA binding. ¹Bands corresponding to the molecular mass of F11 and F12 were also able to bind to the heparin column. ²RNA binding was detected in solution but not by Northwestern-blot analysis. ³Weak RNA binding was detected by Northwestern-blot analysis. (B) Alignment of FSAP from human (Swiss-Prot accession number Q14520), chimpanzee (GenBank® accession number XP_508042), bovine (Swiss-Prot accession number Q5E9Z2), mouse (Swiss-Prot accession number Q8K0D2), rat (Swiss-Prot accession number Q6L711) and chicken (GenBank® accession number XP_421761) indicates that this region is highly conserved in evolution.