Studies on the sialidoses. Properties of human leucocyte neuraminidases

Hong, Victor; Beauregard, Guy; Potier, Michel; Bélisle, M.; Mameli, L.; Gatti, Rosanna; Durand, P.

doi:10.1016/0005-2744(80)90143-6

Cited by 35 publications

(7 citation statements)

References 49 publications

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“…Therefore, cell death with membrane permeabilization would be the expected mechanism for CHO sialidase release into the culture medium (58). Besides, despite controversial results concerning the instability of mammalian sialidase enzymes in cell lysate (59,60), it has been more recently demonstrated that CHO cells possess stable sialidase activity at pH 7 (33), and several publications have reported progressive loss of terminal sialic acid of different recombinant glycoproteins produced by CHO cells as a consequence of increasing extracellular sialidase activity during batch cultivations (56, 61, 62).…”

Section: Profile Consistency Of the Desialylated N-linked Glycans Of mentioning

confidence: 98%

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

et al. 2004

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Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.

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Section: Profile Consistency Of the Desialylated N-linked Glycans Of mentioning

confidence: 98%

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

et al. 2004

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“…The current literature data for mammalian sialidase activities do not strongly support the probability for extracellular hydrolysis of sialic acid from glycoprotein oligosaccharides in conjunction with cell culture. Mammalian sialidase enzymes have been reported to be extremely unstable in cell lysates (Conzelmann and Sandhoff, 1987;Potier et al, 1979a;Den Tandt and Leroy, 1980;Nguyen Hong et al, 1980;Hiraiwa et al, 1987), and most have acidic pH optimums with little activity near pH 7 (Warner and O'Brien, 1979;Spaltro and Alhadeff, 1984;Schauer and Wember, 1984;Sato, 1989;Potier et al, 1979b). Sialidase activity has recently been demonstrated in CHO detergent extracts at pH 6.5 using the artificial substrate 2 ' -( 4 -m e t h y l u m b e l l l ) -~-~~-a c e t y l n e uic acid (4MU-NeuAc) (Potvin and Stanley, 1991).…”

Section: Introductionmentioning

confidence: 99%

Glycosidase Activities in Chinese Hamster Ovary Cell Lysate and Cell Culture Supernatant

Gramer

Goochee

1993

Biotechnology Progress

137

102

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To probe the potential for extracellular degradation of glycoprotein oligosaccharides in conjunction with Chinese hamster ovary (CHO) cell culture, an initial characterization of several CHO cell glycosidases was performed using 4-methylumbelliferyl substrates. CHO cell lysates contained sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities with pH optimums near 5.5, 4, 6, and 6.5, respectively. These glycosidase activities were also present in cell-free supernatant samples from commercial CHO cell cultures. The sialidase activity was further characterized. In contrast to previous reports concerning mammalian sialidases, the sialidase activity in CHO cell lysate retained considerable activity at pH 7 and was very stable, with a half-life of 57 h at 37 degrees C. Both the Km and Vmax of CHO lysate sialidase for 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) varied with pH, and this activity was competitively inhibited by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid and by free N-acetylneuraminic acid. The kinetic characteristics and pH-activity profiles of the CHO cell lysate and cell culture supernatant sialidase activities were essentially identical, and both released sialic acid from the glycoprotein fetuin at pH 7.5. These results suggest that the oligosaccharides of glycoproteins secreted by CHO cells can potentially be modified extracellularly by sialidase under culture conditions which promote the release and extracellular accumulation of this enzyme.

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“…Our studies thus far have shown very little difference in the target size of human or rodent forms of the enzymes studied, with the exception of f,-glucuronidase and arylsulphatase A. Others have reported major size differences between human leucocyte (>200kDa) and rat liver (56kDa) forms of lysosomal neuraminidases (Nguyen Hong et al, 1980;Beauregard & Potier, 1982) with the use of this technique. One of the major advantages of the radiation inactivation technique is that molecular masses can be obtained on whole-cell preparations without any need for enzyme purification.…”

Section: Discussionmentioning

confidence: 60%

Functional lysosomal hydrolase size as determined by radiation inactivation analysis

Dawson¹,

Ellory²

1985

Biochemical Journal

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Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.

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Studies on the sialidoses. Properties of human leucocyte neuraminidases

Cited by 35 publications

References 49 publications

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

Glycosidase Activities in Chinese Hamster Ovary Cell Lysate and Cell Culture Supernatant

Functional lysosomal hydrolase size as determined by radiation inactivation analysis

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