Residues essential for plasminogen binding by the cation-independent mannose 6-phosphate receptor

Abstract

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that binds diverse intracellular and extracellular ligands with high affinity. The CI-MPR is a receptor for plasminogen, and this interaction can be inhibited by lysine analogues. To characterize the molecular basis for this interaction, surface plasmon resonance (SPR) analyses were performed using truncated forms of the CI-MPR and plasminogen. The results show that the N-terminal region of the CI-MPR containing domains 1 and 2, but not domain 1 alone, of the receptor's 15-domain extracytoplasmic region binds plasminogen (K(d) = 5 +/- 1 nM) with an affinity similar to that of the full-length receptor (K(d) = 20 +/- 6 nM). In addition to its C-terminal serine protease domain, plasminogen contains lysine binding sites (LBS), which are located within each of its five kringle domains, except kringle 3. We show that kringles 1-4, but not kringles 1-3, bind the CI-MPR, indicating an essential role for the LBS in kringle 4 of plasminogen. To identify the lysine residue(s) of the CI-MPR that serve(s) as an essential determinant for recognition by the LBS of plasminogen, site-directed mutagenesis studies were carried out using a construct encoding the N-terminal three domains of the CI-MPR (Dom1-3His) which contains both a mannose 6-phosphate (Man-6-P) and plasminogen binding site. The results demonstrate two lysine residues (Lys53 located in domain 1 and Lys125 located in the loop connecting domains 1 and 2) of the CI-MPR are key determinants for plasminogen binding but are not required for Man-6-P binding.

FIGURE 1. MPR constructs

(A) Schematic diagram of sCI-MPR, Dom1-3His, Dom1-2His, and Dom1His constructs. Based on its size, the sCI-MPR is predicted to contain most of the extracellular region (i.e., extracellular domains 1-15). However, the site of cleavage (red arrow) is not known and could occur within domain 15. The small white squares indicate potential N-glycosylation sites. The location of the ligand binding sites is shown. Domains 1 and 2 are outlined in green since the presence of these two domains enhances the affinity of domain 3 for lysosomal enzymes by ~1000-fold (24). (B) SDS polyacrylamide gel of sCI-MPR purified from bovine serum and Dom1-3His, Dom1-2His, and Dom1His constructs purified from the medium of transformed P. pastoris yeast. The proteins are visualized by silver staining. The multiple bands observed for Dom1-3His are due to differences in the utilization of the three N-glycosylation sites as determined by enzymatic deglycosylation using endo-β-N-acetylglucosaminidase H digestion (data not shown).

FIGURE 2. Sensorgrams measuring the interaction between the MPRs (sCI-MPR or Dom1-3His) and IGF-II or a lysosomal enzyme

The sCI-MPR and Dom1-3His proteins were coupled to separate flow cells of a CM5 sensor chip, with a final coupling level of 2600 and 1000 response units (RU), respectively. Increasing concentrations of IGF-II or the lysosomal enzyme, β-glucuronidase, were injected onto the chip. (A) Sensorgrams show the interaction between the sCI-MPR and IGF-II (1, 2.5, 5, 10, 25, 50, and 100 nM). (B) Sensorgrams depict the interaction between β-glucuronidase (0.5, 1, 2.5, 5, 10, 25, 50, and 100 nM) and the sCI-MPR or (C) Dom1-3His. The insets in panels B and C are sensorgrams showing the interaction between β-glucuronidase (50 nM) and sCI-MPR or Dom1-3His, respectively, in the absence or presence of 10 mM Man-6-P.

FIGURE 3. 6-aminohexanoic acid (AHA) inhibits the interaction between Glu-plasminogen and the sCI-MPR or Dom1-3His

SPR analyses were used to estimate the half-maximal inhibition of sCI-MPR (λ) and Dom1-3His (μ) binding to Glu-plasminogen by AHA. Briefly, Glu-plasminogen was diluted to 25 nM, mixed with increasing concentrations of AHA (0, 0.1, 0.5, 1, 5, 10, 25, 50, 100, 250, 500, 750, 1000, and 1500 μM), and the samples were injected onto the coupled flow cells. The response at equilibrium (Req) was determined and plotted against the log of the AHA concentration. The K_i (sCI-MPR K_i = 29 ± 1.3 μM; Dom1-3His K_i = 26 ± 1.0 μM) was determined by non-linear regression using Sigmaplot (version 10.0, Systat Software, Inc.).

FIGURE 4. The conformation of Glu-plasminogen influences its interaction with the CI-MPR

Aliquots containing 2.5, 5, 7.5, 10, 15, 20, 30, 40, 60, 100, and 250 nM bovine Glu-plasminogen in HBA buffer containing 150 mM sodium acetate, pH 7.4 (μ) or HBS buffer containing 150 mM NaCl, pH 7.4 (λ), or aliquots containing 1, 2.5, 5, 10, 15, 25, 50, 75, 100, and 250 nM Glu-plasminogen in HBA buffer with 50 mM benzamidine (τ) were injected onto a Dom1-3His-coupled CM5 sensor chip. The response at equilibrium (Req) was determined and plotted against the concentration of Glu-plasminogen ([Plg]). The data were fit by non-linear regression using a two-site saturation binding model.

FIGURE 5. Location of lysine residues in domains 1-3 of the CI-MPR

A ribbon diagram overlayed with a transparent surface model of the crystal structure of domains 1-3 of the CI-MPR (PDB accession number 1SZ0) is shown. The view in panel B is rotated 180° from that shown in panel A. The side chains of all lysine residues in domain 1 (green) and domain 2 (orange) are shown in blue, and the lysine residues that have been mutated are indicated in red. Arg118 is highlighted in purple. The bound Man-6-P in domain 3 (gray) is also shown (yellow ball-and-stick). The figure was generated using PyMOL (60).

FIGURE 6. Domain 3 of the CI-MPR is not required for the receptor’s interaction with Glu-plasminogen

Aliquots containing 10, 25, 50, 75, 100, 150, and 250 nM bovine Glu-plasminogen in HBA buffer containing 150 mM sodium acetate, pH 7.4 were injected onto CM5 sensor chips containing coupled Dom1-3His (μ), Dom1-2His (τ), or Dom1His (λ). The response at equilibrium (Req) was determined and plotted against the concentration of Glu-plasminogen ([Plg]). The data were fit by non-linear regression using a two-site saturation binding model.

FIGURE 7. SPR analyses of wild-type and mutant Dom1-3His constructs interaction with Glu-plasminogen or β-glucuronidase

Similar amounts of Dom1-3His, K53A, K125A, and K132A were immobilized on the surface of a CM5 sensor chip and increasing concentrations of Glu-plasminogen or β-glucuronidase were injected over the MPR and reference flow cells at a flow rate of 40 μl/min. Shown are representative sensorgrams for (A) Dom1-3His, (B) K53A, (C) K125A, and (D) K132A at three concentrations (10, 40, and 120 nM) of Glu-plasminogen (red lines) or β-glucuronidase (blue lines), with the exception of K132A in which sensorgrams at concentrations of 10, 50 and 100 nM β-glucuronidase are shown.

FIGURE 8. Lys53 and Lys125 of the CI-MPR are required for the receptor’s interaction with Glu-plasminogen

Aliquots containing 10, 25, 50, 75, 100, 150, and 250 nM bovine Glu-plasminogen in HBA buffer containing 150 mM sodium acetate, pH 7.4 were injected onto CM5 sensor chips containing coupled Dom1-3His (μ), K53A (▼;), or K125A (λ). The response at equilibrium (Req) was determined from the resulting sensorgrams and plotted against the concentration of Glu-plasminogen ([Plg]). The data were fit by non-linear regression using a two-site saturation binding model.