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Selection against glycosylation in ruminant pancreatic ribonucleases by replacements in the ancestral carbohydrate attachment site.

Gautschi M, Beintema JJ - Biochem. Genet. (2008)

Bottom Line: A hypothesis, proposed 25 years ago, that there is selection against glycosylation in ruminant pancreatic ribonucleases by replacement of methionine to leucine in the ancestral carbohydrate attachment site Asn-Met-Thr at residues 34-36, was experimentally confirmed.The replacement of leucine at position 35 by methionine in bovine ribonuclease resulted in a three-fold relative increase in glycosylation when expressed in Chinese hamster ovary cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biophysics, Schafmattstrasse 20, ETH-Hönggerberg, HPKE 14, 8093, Zurich, Switzerland.

ABSTRACT
A hypothesis, proposed 25 years ago, that there is selection against glycosylation in ruminant pancreatic ribonucleases by replacement of methionine to leucine in the ancestral carbohydrate attachment site Asn-Met-Thr at residues 34-36, was experimentally confirmed. The replacement of leucine at position 35 by methionine in bovine ribonuclease resulted in a three-fold relative increase in glycosylation when expressed in Chinese hamster ovary cells.

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(a) One representative example of three independent pulse/chase experiments. Pulse (3 min/0.25 mCi/ml 35S met/cys), direct lysis (0 min), or chase (60 min) of wild type (wt) and L35M bovine RNase in Chinese hamster ovary cells. After immunoprecipitation of RNase molecules with RNase antibodies from the lysate (cells) and medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. Note: The cell-associated RNase B at 60 min is composed of Golgi forms (slow-migrating) and endoplasmic reticulum forms (fast-migrating) (bold bar). (b) After quantification of all three pulse chases using ImageQuant, the mean values of ratio of RNase B to RNase A were plotted using Kaleidograph. Standard deviations are given by the error bars
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Fig2: (a) One representative example of three independent pulse/chase experiments. Pulse (3 min/0.25 mCi/ml 35S met/cys), direct lysis (0 min), or chase (60 min) of wild type (wt) and L35M bovine RNase in Chinese hamster ovary cells. After immunoprecipitation of RNase molecules with RNase antibodies from the lysate (cells) and medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. Note: The cell-associated RNase B at 60 min is composed of Golgi forms (slow-migrating) and endoplasmic reticulum forms (fast-migrating) (bold bar). (b) After quantification of all three pulse chases using ImageQuant, the mean values of ratio of RNase B to RNase A were plotted using Kaleidograph. Standard deviations are given by the error bars

Mentions: As Asn-linked glycosylation in the endoplasmic reticulum mainly occurs on the nascent polypeptide chain, only the primary structure of a carbohydrate attachment site will be of influence on the extent of its glycosylation. Therefore, we predicted that a replacement of the Leu in the Asn–Leu–Thr sequence in bovine RNase 1 to a Met (L35M) will enhance glycosylation (Beintema and Lenstra 1982). When we made this prediction 25 years ago, it was not yet possible to test this experimentally. However, now it is possible to construct a mutant with this replacement and express it in parallel to the wild type in Chinese hamster ovary (CHO) cells and to study the amount of carbohydrate attached in the product obtained (Deprez et al. 2005). As shown in Fig. 2, the ratio of glycosylated (RNase B) to unglycosylated (RNase A) of wild type bovine RNase in CHO cells is about 1, while in vivo in bovine pancreas this ratio is less than 0.2 (Hirs et al. 1953), indicating a clear cell type difference. In the L35M mutant in CHO cells there is indeed a three-fold relative increase in glycosylation. The L35M mutant behaves like wild type in CHO cells, as shown by the stable ratio of glycosylated to unglycosylated forms over 60 min and by the amounts of secreted (medium) versus cell-associated (cells) forms after 60 min (Fig. 2). This indicates that the L35M mutant is unchanged in respect to folding and stability, but with enhanced glycosylation.Fig. 2


Selection against glycosylation in ruminant pancreatic ribonucleases by replacements in the ancestral carbohydrate attachment site.

Gautschi M, Beintema JJ - Biochem. Genet. (2008)

(a) One representative example of three independent pulse/chase experiments. Pulse (3 min/0.25 mCi/ml 35S met/cys), direct lysis (0 min), or chase (60 min) of wild type (wt) and L35M bovine RNase in Chinese hamster ovary cells. After immunoprecipitation of RNase molecules with RNase antibodies from the lysate (cells) and medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. Note: The cell-associated RNase B at 60 min is composed of Golgi forms (slow-migrating) and endoplasmic reticulum forms (fast-migrating) (bold bar). (b) After quantification of all three pulse chases using ImageQuant, the mean values of ratio of RNase B to RNase A were plotted using Kaleidograph. Standard deviations are given by the error bars
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480611&req=5

Fig2: (a) One representative example of three independent pulse/chase experiments. Pulse (3 min/0.25 mCi/ml 35S met/cys), direct lysis (0 min), or chase (60 min) of wild type (wt) and L35M bovine RNase in Chinese hamster ovary cells. After immunoprecipitation of RNase molecules with RNase antibodies from the lysate (cells) and medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. Note: The cell-associated RNase B at 60 min is composed of Golgi forms (slow-migrating) and endoplasmic reticulum forms (fast-migrating) (bold bar). (b) After quantification of all three pulse chases using ImageQuant, the mean values of ratio of RNase B to RNase A were plotted using Kaleidograph. Standard deviations are given by the error bars
Mentions: As Asn-linked glycosylation in the endoplasmic reticulum mainly occurs on the nascent polypeptide chain, only the primary structure of a carbohydrate attachment site will be of influence on the extent of its glycosylation. Therefore, we predicted that a replacement of the Leu in the Asn–Leu–Thr sequence in bovine RNase 1 to a Met (L35M) will enhance glycosylation (Beintema and Lenstra 1982). When we made this prediction 25 years ago, it was not yet possible to test this experimentally. However, now it is possible to construct a mutant with this replacement and express it in parallel to the wild type in Chinese hamster ovary (CHO) cells and to study the amount of carbohydrate attached in the product obtained (Deprez et al. 2005). As shown in Fig. 2, the ratio of glycosylated (RNase B) to unglycosylated (RNase A) of wild type bovine RNase in CHO cells is about 1, while in vivo in bovine pancreas this ratio is less than 0.2 (Hirs et al. 1953), indicating a clear cell type difference. In the L35M mutant in CHO cells there is indeed a three-fold relative increase in glycosylation. The L35M mutant behaves like wild type in CHO cells, as shown by the stable ratio of glycosylated to unglycosylated forms over 60 min and by the amounts of secreted (medium) versus cell-associated (cells) forms after 60 min (Fig. 2). This indicates that the L35M mutant is unchanged in respect to folding and stability, but with enhanced glycosylation.Fig. 2

Bottom Line: A hypothesis, proposed 25 years ago, that there is selection against glycosylation in ruminant pancreatic ribonucleases by replacement of methionine to leucine in the ancestral carbohydrate attachment site Asn-Met-Thr at residues 34-36, was experimentally confirmed.The replacement of leucine at position 35 by methionine in bovine ribonuclease resulted in a three-fold relative increase in glycosylation when expressed in Chinese hamster ovary cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biophysics, Schafmattstrasse 20, ETH-Hönggerberg, HPKE 14, 8093, Zurich, Switzerland.

ABSTRACT
A hypothesis, proposed 25 years ago, that there is selection against glycosylation in ruminant pancreatic ribonucleases by replacement of methionine to leucine in the ancestral carbohydrate attachment site Asn-Met-Thr at residues 34-36, was experimentally confirmed. The replacement of leucine at position 35 by methionine in bovine ribonuclease resulted in a three-fold relative increase in glycosylation when expressed in Chinese hamster ovary cells.

Show MeSH
Related in: MedlinePlus