Biochemical and Biophysical Research Communications
Myelin basic protein stimulates plasminogen activation via tissue plasminogen activator following binding to independent l-lysine-containing domains
Graphical abstract
t-PA binds to Lys91 in the MBP NH2-terminal region and Pg binds to Lys122 in the MBP COOH-terminal region. This proximity promotes the activation of Pg by t-PA. The Pm generated may proteolyze MBP, displacing Pm from the surface which then is inhibited by α2-antiplasmin in the vicinity.
Introduction
Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) are serine proteases commonly associated with fibrinolysis [1]; however, they also play important roles in the central nervous system (CNS) [2], [3]. t-PA is primarily involved in synaptic formation and plasticity [4] via mechanisms both dependent [5], [6] and independent [7], [8], [9] of its proteinase activity. In addition to their fibrinolytic functions [10], both t-PA and Pg are also involved in the neuroinflammation observed in patients suffering from pathologies, such as multiple sclerosis (MS) and encephalitis [11], in which demyelination and axonal damage are responsible for neurological deficits [12], [13].
Pg activation in the normal brain is tightly regulated, possibly because it is neurotoxic [10]; however, plasmin (Pm) may play a role in the generation of long-term potentiation (LTP) in the rat hippocampus [14]. Furthermore, a recent report suggests that Pm, along with t-PA, is involved in the blood-brain barrier (BBB) disruption that occurs during t-PA-induced thrombolysis in ischemic stroke [15]. Both t-PA and Pg are expressed in neurons, astrocytes and microglia [10]. Oligodendrocytes are responsible for the production and maintenance of myelin, the specialized lipid membrane that encases the axons of all neurons in the brain [16]. Myelin is composed of lipids and two proteins, myelin basic protein (MBP) and proteolipid protein [16]. The integrity of the myelin sheath may be disrupted by Pg conversion to Pm by t-PA because Pm may hydrolize MBP [17].
We found that both t-PA and Pg bind to MBP with high affinity, and that Pg activation by t-PA is stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the MBP NH2-terminal region, Asp82-Pro99, and the binding of Pg via a lysine-dependent mechanism to the MBP COOH-terminal region, Leu109-Gly126.
Section snippets
Materials
Culture media were purchased from Life Technologies (Gaithersburg, MD). The chromogenic substrates V-L-K-pNA (S-2251) and I-P-R-pNA (S-2288) were purchased from Diapharma (West Chester, OH). MBP peptides D82ENPVVHFFKNIVTPRTP99 (Asp82-Pro99), T98PPPSQGKGRGLSLSRFS115 (Thr98-Ser115), L109SLSRFSWGAEGQKPGFG126 (Leu109-Gly126) and ARGQGPYFSWGGFSEKIG (scrambled L109-G126) were obtained from Bachem Americas, Inc. (Torrance, CA). Tranexamic acid (TXA) was purchased from Sigma (St. Louis, MO). The
Binding of Pg and t-PA to immobilized MBP
Pg binds to immobilized MBP in a dose-dependent manner (Fig. 1A) with high affinity (Kd = 50.26 ± 8.38 nM). Similarly, t-PA binds to MBP in a dose-dependent manner (Fig. 1B) with high affinity (Kd = 49.45 ± 8.32 nM). The binding of Pg to MBP is not inhibited by t-PA (Fig. 1C) and the binding of t-PA to MBP is not inhibited by Pg (Fig. 1D), suggesting that both proteins bind to independent sites in MBP. Binding of both Pg and t-PA to immobilized MBP is inhibited by TXA (Fig. 2A and B,
Discussion
The Pg activator system plays an important role in the CNS, including processes of neuronal migration, neurite outgrowth, and neuronal plasticity [24], [25]. The Pg activator, t-PA, may also participate in several neuropathological conditions, such as cerebral ischemia, Alzheimer's disease and multiple sclerosis [25]. t-PA and its substrate Pg are expressed in neurons, astrocytes and microglia [10].
Pm activity in the normal brain is tightly regulated [10]. We hypothesize that MBP, a prominent
Acknowledgements
This study was supported by a grant from Fondo Nacional de Desarrollo Cientίfico y Tecnolόgico de Chile, FONDECYT No 1130451.
References (33)
- et al.
The fibrinolytic system in man
Crit. Rev. Oncol. Hematol.
(1986) - et al.
tPA in the injured central nervous system: different scenarios starring the same actor?
Neuropharmacology
(2012) - et al.
Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway
Neuron
(1998) - et al.
Tissue plasminogen activator increases canine endothelial cell proliferation rate through a plasmin-independent, receptor mediated mechanism
J. Surg. Res.
(1996) - et al.
Abnormal morphology of myelin and axon pathology in murine models of multiple sclerosis
Neurochem. Int.
(2015) - et al.
A role for the plasminogen activator system in inflammation and neurodegeneration in the central nervous system during experimental allergic encephalomyelitis
Am. J. Pathol.
(2005) - et al.
Possible involvement of plasmin in long-term potentiation of rat hippocampal slices
Brain Res.
(1996) - et al.
Central nervous system myelin: structure, synthesis and assembly
Trends Cell Biol.
(2011) - et al.
Plasminogen structural domains exhibit different functions when associated with cell surface GRP78 or the voltage-dependent anion channel
J. Biol. Chem.
(2007) - et al.
Kinetics of activation of human plasminogen by different activators species at pH 7.4 and 37°C
J. Biol. Chem.
(1980)