Abstract
Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 x 10(7) M-1, as determined by surface plasmon resonance, a value close to that (1.6 x 10(7) M-1) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.
Highlights
The plasma glycoprotein plasminogen (Pg)1 is a single-chain 92-kDa precursor for the broad spectrum serine proteinase plasmin [1, 2]
In this paper we show that K2, binding very weakly to free lysine or lysine analogues [13, 14], mediates high affinity binding of Pg to PAM and its internal a repeats
To map the region in Pg (Fig. 1A) mediating the interaction with PAM (Fig. 1B), we first measured the ability of radiolabeled elastase-generated human Pg fragments to bind to three group A streptocccal strains expressing different M proteins: the PAM-expressing strain AP53, the strain JRS145(Arp) that expresses the IgA-binding protein Arp4, and the strain JRS145(Arp/a1a2) that expresses a chimeric surface protein in which a 36-amino acid-long sequence overlapping the IgA-binding site of Arp4 has been replaced with the combined a1a2 repeats of PAM
Summary
The plasma glycoprotein plasminogen (Pg) is a single-chain 92-kDa precursor for the broad spectrum serine proteinase plasmin [1, 2] (see Fig. 1A). The activable serine proteinase domain is located in the COOH-terminal third of Pg. The NH2-terminal two-thirds of Pg contains an 8-kDa preactivation peptide and five characteristic kringle domains (K1–K5), each ϳ9 kDa. The kringles mediate interactions with multiple ligands, including fibrin, the primary target of Pg, and ␣2-plasmin inhibitor, its principal regulator [1, 2]. The recognition events depend upon interactions between lysine-binding sites in the kringles and exposed COOH-terminal lysines in the ligands. Lysine analogues, such as 6-aminohexanoic acid (6-AHA), mimic COOH-terminal lysines in the interaction with kringles and the structural basis of the interactions between some kringles, K1 and K4, and 6-AHA has been disclosed (9 –12). In this paper we show that K2, binding very weakly to free lysine or lysine analogues [13, 14], mediates high affinity binding of Pg to PAM and its internal a repeats
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