Freeze-stable sialidase activity in human leucocytes: substrate specificity, inhibitor susceptibility, detergent requirements and subcellular localization

Abstract

Human leucocytes contain a freeze-stable sialidase (neuraminidase; EC 3.2.1.18) activity in addition to the better-characterized lysosomal freeze-labile enzyme. In order to discriminate between the sialidase activities detected with the synthetic fluorimetric substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (MU-Neu5Ac), different tritiated sialoglycoconjugate substrates were prepared. Using this sensitive radioactive assay system, leucocyte sialidase activity towards glycoproteins was shown to be labile to repeated freeze-thawing, but a Triton-stimulated activity towards gangliosides was entirely freeze-stable. Assay conditions were optimized for this freeze-stable ganglioside sialidase activity. Subcellular fractionation of mononuclear leucocytes (MNLs) on Percoll-density gradients showed that this ganglioside sialidase activity was entirely associated with the plasma membrane. Study of the detergent requirements showed that MNLs also demonstrated ganglioside sialidase activity when sodium cholate was present in place of Triton. Cholate-stimulated ganglioside sialidase activity was found to be entirely freeze-stable and localized at the plasma membrane. Studies on whole homogenates of MNLs demonstrated that the Triton-stimulated and cholate-stimulated activities showed similar acidic pH optima at < or = 3.9 and were both strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and Cu2+, but not by free N-acetylneuraminic acid, N-(4-nitrophenyl)oxamic acid or heparan sulphate. These results suggest that human MNLs contain, in addition to the lysosomal freeze-labile sialidase, a single sialidase activity which is freeze-stable, ganglioside-specific, plasma membrane-associated and stimulated both by Triton and by cholate.

Improved storage of erythrocytes by prior leukodepletion: Flow cytometric evaluation of stored erythrocytes

“…With improvements over the last 20 years in the techniques to prepare leukodepleted cellular blood components, it has been possible to study the potential damage that could be wrought by the many degradative enzymes that are released by the progressive lysis of leukocytes on storage. These are mainly proteases that are responsible for hemolysis (13, 15, 22), because stored RBCs lack the protective effect of plasma which contains an array of antiproteases (33) and sialidases that have been identified in lytic products of leukocytes (9–11, 14) and to which the cause of the premature senescence of stored RBCs and the loss of viability of these cells could be attributed.…”

“…First, apoptosis occurs in white cells within the first 48–72 h of storage, leading to the formation of membrane fragments that adhere to RBCs and initiate erythrophagocytosis (9–11). Second, by the same mechanism, active enzymes, including neuraminidases, are released in the storage medium (12–15). Third, many authors have reported the advantages of leukodepletion prior to storage of RBCs in preventing the deleterious effects observed with nonleukodepleted RBC transfusion (see refs.…”

Bacterial neuraminidase‐mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure

“…7,15 While the escalation in neuraminidase activity observed in sepsis may be attributed to the presence of bacteria in the circulation, 36 neuraminidases released from host cells including platelets, leukocytes, and erythrocytes per se may, in addition, contribute to this effect. 15,34,37 Mounting evidence suggests that increased PS externalization, triggered by a host of bacterial components such as peptidoglycan, lipopeptides, sphingomyelinase, Pseudomonas aeruginosa pyocyanin, and α-hemolysin, shortens erythrocyte lifespan, thus, contributing to sepsis-associated anemia. 17,20,22,29,38 Along these lines, it may be conjectured that enhanced neuraminidase activity in septic patients contributes to aggravated eryptosis and anemia.…”

“…Importantly, both increased plasma levels of SA and blood neuraminidase activity have been reported in septic patients . While the escalation in neuraminidase activity observed in sepsis may be attributed to the presence of bacteria in the circulation, neuraminidases released from host cells including platelets, leukocytes, and erythrocytes per se may, in addition, contribute to this effect . Mounting evidence suggests that increased PS externalization, triggered by a host of bacterial components such as peptidoglycan, lipopeptides, sphingomyelinase, Pseudomonas aeruginosa pyocyanin, and α‐hemolysin, shortens erythrocyte lifespan, thus, contributing to sepsis‐associated anemia .…”

“…The mechanistic overlaps between erythrocyte senescence and eryptosis have remained largely obscure. One clue suggesting a possible intersection of these phenomena comes from a previous study showing that erythrocytes stored under non-leukodepleted conditions display substantial time-dependent increases in PS exposure, which parallels a reduction in surface SA expression, pointing to a possible link between erythrocyte sialylation status and eryptosis30 ; neuraminidases released by the leukocyte population in these conditions may potentially explain the loss of erythrocyte SA 34. Notwithstanding the profound desialylating effect of exogenous bacterial neuraminidase on erythrocytes in our in vitro experiments, the percentage of PS-exposing erythrocytes was ~15% and a similar proportion of erythrocytes displayed hemolysis.…”