Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains

The reticuloendothelial system plays a major role in iron metabolism. Despite this, the manner in which macrophages handle iron remains poorly understood. Mammalian cells utilize transferrin-dependent mechanisms to acquire iron via transferrin receptors 1 and 2 (TfR1 and TfR2) by receptor-mediated endocytosis. Here, we show for the first time that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is localized on human and murine macrophage cell surface. The expression of this surface GAPDH is regulated by the availability of iron in the medium. We further demonstrate that this GAPDH interacts with transferrin and the GAPDHtransferrin complex is subsequently internalized into the early endosomes. Our work sheds new light on the mechanisms involved in regulation of iron, vital for controlling numerous diseases and maintaining normal immune function. Thus, we propose an entirely new avenue for investigation with respect to transferrin uptake and regulation mechanisms in macrophages.Iron is an essential nutrient for all organisms as a constituent of hemoproteins and iron-sulfur proteins. In addition it is also a critical component of functional groups of several proteins involved in vital housekeeping functions. Cells of the immune system require iron for their normal functions such as proliferation, activation, and maturation of lymphocytes (1-5). Iron is also essential for macrophage-mediated cytotoxicity by the production of highly toxic hydroxyl radicals (6, 7). The mononuclear phagocyte system is composed of monocytes, macrophages, and their precursor cells, which play a vital role in iron metabolism by removing effete erythrocytes and recycling iron. These cells also acquire iron via the receptor-mediated uptake of transferrin and the hemoglobin scavenger receptor (8). Practically all extracellular iron circulating in the plasma is bound to transferrin, an abundant protein with high affinity for iron. Two mammalian transferrin receptors TfR1 4 and TfR2 have so far been characterized. Both these receptors are cell surface transmembrane, glycoproteins (9). Unlike TfR1, TfR2 is not regulated by intracellular iron concentrations. This receptor also binds transferrin in manner similar to TfR1, but with a 25-fold lower affinity (10,11,12). Iron uptake from transferrin involves binding to its receptors followed by internalization to the early endosomes. (13, 14). Although TfR-mediated iron uptake is the major pathway for iron acquisition, several studies have indicated that additional mechanisms independent of known TfRs exist; however, these have not been well characterized (15-18). GAPDH was previously considered to be an abundantly present cytosolic protein with a key role in energy metabolism. However, recent evidence has proved that it functions as a moonlighting protein in both prokaryotic and eukaryotic cells, often differentially localized within the cell (19 -21). It is of interest to note that in Staphylococcus aureus, cell wall-associated GAPDH had previously been identified as a trans...

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“…Experimental procedure was essentially as described previously (28,29). Streptavidin was captured on biotin cuvettes.…”

Section: Analysis Of Protein-protein Interactions Using Resonant Mirrmentioning

confidence: 99%

The Macrophage Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase Is a Novel Transferrin Receptor

Raje¹,

Kumar²,

Harle

et al. 2007

Journal of Biological Chemistry

124

111

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“…The ␤ domain provides no direct contact sites with the plasmin active-site moiety, yet it is required to dock plasminogen via a kringle-binding surface-exposed hairpin loop (termed the 250-loop) between residues 251 and 262 ( Fig. 1A) (1,18). In contrast, a region between amino acids 144 and 218 which spans the second structural loop of the ␤ domain (170-loop) (Fig.…”

mentioning

confidence: 99%

“…Studies on SK structure and function have focused entirely on therapeutic SK from a nonpathogenic group C streptococcus and have not considered the role of sequence polymorphism of the ␤ domain on plasminogen activation (1,2,9,18,19). In an attempt to begin addressing the biological importance of structural heterogeneity in SK, we tested whether the removal or exchange of the major polymorphic region in the ␤ domain had any effect on the activation of plasminogen.…”

mentioning

confidence: 99%

Structural Diversity of Streptokinase and Activation of Human Plasminogen

Lizano

Johnston

2005

Infect Immun

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The ␤ domain of streptokinase is required for plasminogen activation and contains a region of sequence diversity associated with infection and disease in group A streptococci. We report that mutagenesis of this polymorphic region does not alter plasminogen activation, which suggests an alternative function for this molecular motif in streptococcal disease.Streptokinase (SK) is a plasminogen activator secreted by group A, C, and G streptococci. SK contributes to streptococcal virulence by generating plasmin, which leads to bacterial spread from a primary focus of infection by causing fibrinolysis and degradation of extracellular matrix and basement membrane components (6, 16). Plasmin also induces inflammation via complement activation, which may play a role in postinfectious diseases, such as glomerulonephritis (12).The extent to which SK is involved in streptococcal pathogenesis may depend on structural differences among SKs. SK is a 414-amino-acid (aa) protein composed of three structural domains: ␣ (aa 1 to 150), ␤ (aa 151 to 287), and ␥ (aa 288 to 414) (19). The highly conserved (Ն85% sequence identity) ␣ and ␥ domains provide most of the contact sites with the plasmin moiety (2, 19) and exhibit a synergistic effect on plasminogen activation (9). The ␤ domain provides no direct contact sites with the plasmin active-site moiety, yet it is required to dock plasminogen via a kringle-binding surface-exposed hairpin loop (termed the 250-loop) between residues 251 and 262 ( Fig. 1A) (1, 18). In contrast, a region between amino acids 144 and 218 which spans the second structural loop of the ␤ domain (170-loop) (Fig. 1A) is the major focus of sequence heterogeneity (4), yet its function is unknown. The side chains of nonconserved amino acids within this heterogenous loop are oriented towards the surface, away from the side that faces the plasmin moiety of the activator complex (18).This suggests that SK polymorphism may not be directly engaged in plasminogen binding and activation but instead may determine biological properties related to disease. Previous studies have linked sequence patterns within this polymorphic motif to specific strains of group A streptococci (GAS) that cause acute poststreptococcal glomerulonephritis (APSGN) (10). Also, phylogenetic analysis of ␤-domain sequences reveals a strong linkage disequilibrium (P Ͻ Ͻ 0.01) with plasminogen-binding group A streptococcal M protein (PAM) in strains that exhibit tissue tropism to the skin (5). Considering the fact that both SK and PAM cooperate to enhance streptococcal virulence through plasminogen activation (15), the specific association between ␤-domain polymorphism and tissue tropism further supports a role of this motif in disease.Despite these associations, the biological significance of sequence polymorphism in SK remains unknown. Studies on SK structure and function have focused entirely on therapeutic SK from a nonpathogenic group C streptococcus and have not considered the role of sequence polymorphism of the ␤ domain on plasminogen activation...

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“…In addition, the newly formed SAK Lys 10 N terminus appears to interact with a regulatory kringle domain of the substrate Pg (40). Although similar structural evidence is missing for the SK⅐Pg/Pm complexes, the results of recent investigations demonstrate that Pg substrate binding is mediated by expression of a new exosite on the SK⅐Pm complex (41) and is facilitated by interaction of specific lysine residues of the bacterial cofactor with kringle domain(s) of the substrate Pg molecule (42)(43)(44)(45).…”

Section: Discussionmentioning

confidence: 91%