The role of sialylation in kidney biology is not fully understood. The synthesis of sialoglycoconjugates, which form the outermost structures of animal cells, requires CMP-sialic acid, which is a product of the nuclear enzyme CMAS. We used a knock-in strategy to create a mouse with point mutations in the canonical nuclear localization signal of CMAS, which relocated the enzyme to the cytoplasm of transfected cells without affecting its activity. Although insufficient to prevent nuclear entry in mice, the mutation led to a drastically reduced concentration of nuclear-expressed enzyme. Mice homozygous for the mutation died from kidney failure within 72 hours after birth. The Cmas nls mouse exhibited podocyte foot process effacement, absence of slit diaphragms, and massive proteinuria, recapitulating features of nephrinknockout mice and of patients with Finnish-type congenital nephrotic syndrome. Although the Cmas nls mouse displayed normal sialylation in all organs including kidney, a critical shortage of CMP-sialic acid prevented sialylation of nephrin and podocalyxin in the maturing podocyte where it is required during the formation of foot processes. Accordingly, the sialylation defects progressed with time and paralleled the morphologic changes. In summary, sialylation is critical during the development of the glomerular filtration barrier and required for the proper function of nephrin. Whether altered sialylation impairs nephrin function in human disease requires further study.

These data demonstrate that r-hirudin can be used successfully as an alternative anticoagulant to heparin during cardiac operations including cardiopulmonary bypass. The better preservation of platelet function suggests that r-hirudin may reduce the postoperative risk of bleeding.

Background: Addition of sialic acid to the nonreducing end of glycoconjugates requires activation by CMP-sialic acid synthetase (CMAS). Results: In zebrafish, we identified two CMAS enzymes that differ in expression pattern, activities, and intracellular localization. Conclusion: Maintenance of two CMAS paralogues is attributed to subfunctionalization. Significance: Unraveling the individual functions of CMAS paralogues helps to elucidate the impact of sialylation in vertebrate development.

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The human pathogens Escherichia coli K1 and Neisseria meningitides serogroup B (NmB) are major causes of bacterial meningitis. These bacteria express capsular polysaccharides that are chemically and immunologically identical to the polysialic acid (polySia) expressed in the host organism. This structural mimicry prevents the production of potentially hazardous autoantibodies, and furthermore, the hydrated, negatively charged capsules interfere with the activation of the humoral immune system. Phages infecting the polySia-encapsulated human pathogen E. coli K1 are equipped with capsule-degrading tail spikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. Stummeyer et al. have recently reported the X-ray crystal structure of the polySia-degrading endosialidase of bacteriophage FK1F.[1] The endosialidase assembles into a catalytic trimer stabilized by a triple b-helix, and its active site differs markedly from that of exosialidases; this indicates an endosialidase-specific substratebinding mode and catalytic mechanism. However, only a dimeric sialic acid fragment was identified in the binding domain of endoNF when the crystal was soaked with polysialic pentamer (DP5; DP = degree of polymerization), and it remains unclear how polySia (DP5) interacts with endoNF.To better understand how this important enzyme interacts with its substrate, we determined the binding epitope and bound conformation of the a2,8-linked penta-polysialic acid DP5 in complex with endoNF by means of preliminary STD [2,3] and trNOE [4] NMR spectroscopy.To determine the binding epitope, STD NMR experiments were performed using 4.9 mm endoNF (WT) in the presence of DP5 with a 50-fold molecular excess of ligand to protein (50:1). A low temperature of 277 K was employed to prevent cleavage of DP5 during data acquisition. Figure 1 shows the 1 H NMR spectrum (Figure 1 A) and the STD NMR spectrum (Figure 1 B) of DP5 in complex with wild-type endoNF.From Figure 1, it is immediately evident that the enzyme binds DP5. Strong STD NMR signals are observed for the Nacetyl groups (~1.8 ppm), and further analysis of this spectrum (Figure 1 B) suggests that the entire DP5 substrate receives saturation via the protein surface; this indicates that all sialic acid residues of DP5 (Sia1-5) are bound by endoNF. This predicted binding mode is also supported by earlier reports suggesting that the minimal substrate requirement for endosialidase is a polySia DP5.[5] Most striking is the observation, that no STD NMR signal could be obtained for the H3 eq proton of the reducing end of the Sia1 residue resonating at 1.97 ppm (Figure 1 B). This strongly suggests that H3 eq (Sia1) is not in close proximity with the protein surface. Structural comparison of the dimeric sialic acid fragment, as found in the X-ray crystallographic structure, [1] confirms our NMR findings and indicates that whereas Sia1 is in contact with the protein surface, the H3 eq and H3 ax protons of Sia1 are oriented towards the solvent and make no direct c...