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The human α2-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region

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Abstract

The human α2-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn398-Lys452), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys422, Lys429, Lys436, Lys452) are completely and two (Lys406, Lys415) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys452 as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys452 and internal Lys436 of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr445 in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.

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Abbreviations

6-AHA:

6-Aminohexanoic acid

A2PI:

Human α2-plasmin inhibitor

A2PIC:

C-terminal moiety of A2PI (Asn398-Lys452)

rA2PIC:

Recombinant A2PIC (A2PIC/Pro399Ala)

FXa :

Activated coagulation factor X

His-tag:

Peptide MNHKVH6MELGTIEGR

K1:

Kringle 1 of Pgn (Cys84-Cys162), generated as rK1 (Lys78-Glu164)

K2:

Kringle 2 of Pgn (Cys166-Cys243), generated as rK2 (Cys162Thr/Glu163Ser/Glu164-Thr244/Cys169Gly)

K3:

Kringle 3 of Pgn (Cys256-Cys333), generated as rK3 (Thr253-Ser335/Cys297Ser)

rK3mut:

Mutated K3 domain of Pgn (rK3/Lys311Asp)

K4:

Kringle 4 of Pgn (Cys358-Cys435), generated as fragment Val355-Ala440 of Pgn

K5:

Kringle 5 of Pgn (Cys462-Cys541), generated as rK5 (Thr456-Ala543, Pro457Ala)

K1–3:

Kringles 1–3 of Pgn (Cys84-Cys333), generated as fragment Tyr80-Val338 of Pgn

K4–5:

Kringles 4–5 of Pgn (Cys358-Cys541), generated as rK4–5 (Val355-Phe546)

LB Broth:

Luria–Bertani Broth

LBS:

Lysine-binding site

PAN:

Plasminogen N-terminal domain

Pgn:

Human plasminogen

Plm:

Human plasmin

RCL:

Reactive center loop

Serpin:

Serine protease inhibitor

t-AMCHA:

trans-4-(Aminomethyl)cyclohexanecarboxylic acid

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Acknowledgments

We would like to thank PD Dr. A. Walz (Theodor Kocher Institute, University of Bern, Switzerland) for the generation of the synthetic peptides A2PIC(Glu442-Lys452) with and without sulfated Tyr445, Prof. Dr. U. Baumann (University of Bern, Switzerland) for providing the pET22b(+) vector and Prof. L.-O. Hedén (University of Lund, Sweden) for supplying the pPLGKG plasmid. We also thank Mr. U. Kämpfer for expert technical assistance, Mr. Christian Trachsel for his support building the plasminogen model, and Mr. S. Halbherr and Mrs. A. Mori for performing fluorescence measurements during their bachelor theses.

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  1. Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012, Bern, Switzerland

    Simon S. Gerber, Sofia Lejon, Michael Locher & Johann Schaller

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  1. Simon S. Gerber
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  2. Sofia Lejon
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  3. Michael Locher
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  4. Johann Schaller
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Correspondence to Johann Schaller.

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Gerber, S.S., Lejon, S., Locher, M. et al. The human α2-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region. Cell. Mol. Life Sci. 67, 1505–1518 (2010). https://doi.org/10.1007/s00018-010-0264-3

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