MARCKS-related Protein (MRP) Is a Substrate for the Leishmania major Surface Protease Leishmanolysin (gp63)

Abstract

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser 92 and Phe 93, in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.Myristoylated alanine-rich C kinase substrate (MARCKS) 1 and MARCKS-related protein (MRP), also known as Mac-MARCKS, are members of a highly acidic myristoylated family of protein kinase C (PKC) substrates (1, 2). The primary structures of MARCKS and MRP exhibit significant homology, including a highly basic stretch of amino acid residues known as the effector domain (also as the phosphorylation site domain), which contains the serine residues subject to PKC-dependent phosphorylation as well as binding sites for calmodulin and actin. Whereas MARCKS is widely distributed in diverse cell types, MRP is present primarily in brain and reproductive tissue (3, 4) as well as in macrophages, where it was first characterized (5). The biologic functions of MARCKS proteins are unknown. Due to their high effector domain homology, it is also possible that MRP and MARCKS play overlapping roles in some cells. In macrophages, both proteins colocalize in the cytosol in association with components of the actin cytoskeleton (6 -9) and consequently are thought to participate in major cellular responses such as phagocytosis, motility, and membrane trafficking.The expression of MARCKS proteins appears to be highly regulated, and in vitro studies have demonstrated up-or downregulation of MARCKS at both the transcriptional and posttranscriptional levels (5, 10, 11). One mechanism of post-transcriptional regulation involves proteolytic degradation. Spizz and Blackshear (12) recently identified cathepsin B as a cellular MARCKS-cleaving enzyme in fibroblasts. They suggested that cleavage might occur within lysosomes as a result of specific lysosomal targeting sequences identified within the MARCKS primary sequence. At least one c...

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

“…The yield was determined by the Lowry assay (30) by using a correction factor of 1.6 (5). Note that coexpression of MRP with N-myristoyl transferase yields a protein that is correctly myristoylated as judged by the resistance of MRP to N-terminal sequencing by Edmann degradation and by the increase in mass of exact 210 Da measured by mass spectroscopy (unpublished data) (28).…”

“…All mutations in MRP were introduced by using the megaprimer method, which requires only one mutagenic primer per mutation (26,27). We used two general nonmutagenic primers directed against the vector sequences surrounding the insert as described recently (28). Additionally, one mutagenic primer corresponding to the antisense sequence of the mrp gene but containing the sequence 5Ј-CCA-3Ј coding for a tryptophan residue instead of the wild-type sequence was designed for each mutation (29).…”

Subversion Mechanisms by WhichLeishmaniaParasites Can Escape the Host Immune Response: a Signaling Point of View

“…Promastigote LPG has been shown to cause this inhibition (37); however, amastigotes, which lack LPG, can also inhibit PKC in infected human monocytes, indicating the availability of alternative mechanisms (118). LPG-mediated repression may be explained by the observations that it interferes with binding of regulators, including Ca 2ϩ and diacyl glycerol, and can also block PKC membrane insertion (reviewed in refer- (28,29). GIPL, another Leishmania surface glycolipid, may also inactivate PKC, but this process needs further investigation (105).…”

Biological Roles of Peptidases in Trypanosomatids~!2009-11-26~!2010-02-15~!2010-03-18~!

“…On the other hand, the in vivo substrates of gp63 are unknown. Several possible additional roles were suggested for the gp63 molecule when the promastigote is inside the mammalian host: (i) it helps promastigote evasion of complement-mediated lysis, by cleaving C3 to its breakdown products [168] as well as converting C3b to the inactive form iC3b [169], (ii) it enhances phagocytosis of promastigotes through macrophage receptors such as CR3 [169][170][171], (iii) it contains the sequence SRYD that is antigenically related to the RGDS sequence of fibronectin, suggesting a potential interaction of gp63 with macrophage fibronectin receptors [172], (iv) it promotes degradation of host cytosolic MARCKS-related protein [173], (v) it is capable of cleaving surface CD4 and could diminish T cell responses [174] and (vi) it also cleaves intracellular peptides presented by MHC class I molecules [175].…”