Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.

Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.

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Objective-Humans with high expression of apolipoprotein(a) [apo(a)] and high plasma levels of lipoprotein(a) [Lp(a)] are at increased risk for atherosclerosis, but the mechanism is not known. We have previously shown that the KIV 5-8 domain of apo(a) has unique cell-surface binding properties, and naturally occurring fragments of apo(a) encompassing this domain are thought to be atherogenic in humans. To investigate the effect of KIV 5-8 on lipoprotein metabolism and atherosclerosis in vivo, we created several independent lines of liver-targeted KIV 5-8 transgenic mice. Methods and Results-The transgenic mice have plasma apo(a) peptide concentrations that are similar to Lp(a) concentrations in humans at risk for coronary artery disease. Remarkably, the transgenic mice had a 2-to 4-fold increase in cholesterol-rich remnant lipoproteins (RLPs) when fed a cholesterol-rich diet, and a 5-to 20-fold increase in atherosclerosis lesion area in the aortic root. Using an in vivo clearance study, we found only slight differences in the triglyceride and apolipoprotein B secretion rates between the 2 groups of mice, suggesting an RLP clearance defect.Using an isolated perfused mouse liver system, we showed that transgenic livers had a slower rate of RLP removal, which was retarded further when KIV 5-8 , full-length apo(a), or Lp(a) were added to the perfusate. An apo(a) peptide that does not interact with cells, K(IV 2 ) 3 , did not retard RLP removal, and low-density lipoprotein (LDL) had a much smaller effect than Lp(a). Conclusions-We propose that high levels of apo(a)/Lp(a), perhaps acting via a specific cell-surface binding domain, inhibit hepatic clearance of remnants, leading to high plasma levels of RLPs and markedly enhanced atherosclerosis. We speculate that the KIV 5-8 region of apo(a) competes with one or more receptors for remnant clearance in the liver and that this process may represent one mechanism accounting for increased atherosclerosis in humans with high secretion levels of apo(a) [apo(a)] is attached covalently to the apolipoprotein B100 (apoB100) of LDL. 1,2 Apo(a) can also be noncovalently associated with the apoB of very-low-density lipoprotein (VLDL) and remnant lipoproteins (RLPs). [3][4][5] Apo(a) is made up of variable numbers of protein domains called kringles, designated KIV 1 through KIV 10 ( Figure I, available online http://atvb.ahajournals.org). 6 Interest in apo(a)-containing lipoproteins arises from the finding that high plasma levels of Lp(a) are an independent risk factor for atherothrombotic coronary artery disease and stroke. 7,8 Postulated mechanisms include apo(a)-mediated inhibition of plasmin activation on fibrin, stimulation of smooth muscle cell proliferation and migration, increased retention of Lp(a) in the arterial wall, and alterations in endothelial function. 9 -11 Although studies in mice have supported these mechanisms, none has been shown to be directly responsible for the increased risk of coronary artery or cerebrovascular disease in humans..Previous studies ...