An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

Albouz-Abo, Samia; Turton, Richard; Wilson, Jennifer C.; Itzstein, Mark von

doi:10.1016/j.febslet.2004.12.077

Cited by 11 publications

(18 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In agreement with the results obtained for a purified rat liver cytosolic sialidase, 17 adding Cu 2+ (10 mM) significantly decreased the activity of NEU2 to 5%. It is interesting to note that these observations are different from a partially purified recombinant rat cytosolic sialidase whose activity was stimulated by the presence of a divalent cation such as Ca 2+ , Mn 2+ , or Mg 2+ 19. …”

Section: Resultsmentioning

confidence: 75%

See 1 more Smart Citation

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Cao

et al. 2011

Mol. BioSyst.

102

View full text Add to dashboard Cite

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. Low cellular expression level of human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty para-nitrophenol (pNP)-tagged α2–3- or α2–6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.

show abstract

Section: Resultsmentioning

confidence: 75%

“…10,17,18 Plasma membrane NEU3 is believed to be more specific towards gangliosides except for GM1 and GM2. 12,19–21 Similar to NEU2, membrane-associated NEU4 has activity against a broad range of sialylglycoconjugates. 22,23 …”

Section: Introductionmentioning

confidence: 99%

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Cao

et al. 2011

Mol. BioSyst.

102

View full text Add to dashboard Cite

show abstract

“…Itzstein et al reported that hNeu2 require calcium ion for their NA activity, and the NA activity decreases by one-half by the addition of 7 mM copper salt. 28 As shown in Figure 3a, the NA activity of hNeu2 disappeared in the presence of a copper salt even at a very low copper concentration (50 μM CuSO 4 ) in the presence of 4 mM calcium salt. To negate the effect of the copper salt, the effect of a chelating agent for the copper ion, 2-({2-[bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino) acetic acid (EDTA) was evaluated.…”

Section: First Screening Of the Focused Library 3 Prepared By The CLImentioning

confidence: 79%

“…To negate the effect of the copper salt, the effect of a chelating agent for the copper ion, 2-({2-[bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino) acetic acid (EDTA) was evaluated. Although Itzstein et al reported that 1 mM EDTA also decreased the NA activity of hNeu2 by half, 28 addition of 1 equivalent of EDTA recovered the NA activity of hNeu2 completely, and a small excess of EDTA did not affect the activity ( Figure 3). The reported inhibition effect in the presence of 1 mM EDTA might be due to the absence of external addition of calcium ion.…”

Section: First Screening Of the Focused Library 3 Prepared By The CLImentioning

confidence: 86%

See 1 more Smart Citation

Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

Kai

Hinou

Nishimura

2012

Bioorganic & Medicinal Chemistry

View full text Add to dashboard Cite

A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl -sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the K I value with little change in the t 1/2 value of the enzyme activity relative to the basic skeleton (2).

show abstract

Macrocyclic Mechanism‐Based Inhibitor for Neuraminidases

Kai

Hinou

Naruchi

et al. 2012

Chemistry A European J

View full text Add to dashboard Cite

A macrocyclic mechanism-based inhibitor for neuraminidases (NAs) bearing a 2-difluoromethylphenyl aglycone and a linker between the aglycone and C-9 positions of sialic acid was synthesized and evaluated. The macrocyclic structure was designed to keep the aglycone moiety in the active site of the neuraminidase after cleavage of the glycoside bond. When Vibrio chorelae neuraminidase (VCNA) was treated with a similar acyclic derivative in the presence of detergent, the irreversible inhibition property was disabled. In contrast, this macrocyclic compound acted as an irreversible inhibitor for VCNA in the presence of detergent. Inhibition assay for various NAs using this macrocyclic compound revealed that the irreversible inhibition property depends on the k(cat) of the neuraminidase treated. NAs having small k(cat) values, such as Influenza viruses, Clostridium, Trypanosoma cruzi, and Human, were also inhibited irreversibly. However, Salmonella typhimurium NA, which has an extremely high k(cat) , was not affected irreversibly by the inhibitor. Interestingly, in contrast to common k(cat) inhibitors, the irreversibility of inhibition by this macrocyclic compound is inversely proportional to the k(cat) of the target neuraminidase.

show abstract

An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

Cited by 11 publications

References 46 publications

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

Macrocyclic Mechanism‐Based Inhibitor for Neuraminidases

Contact Info

Product

Resources

About