Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein

Chung Ming Tse, Susan A. Levine, C. H Chris Yun, Seema Khurana, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-α-D-galactosaminide (BzαGalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the BzαGalNAc treatment of PS120/NHE2 cells. These results suggest that the 85 kDa protein is an O-glycosylated form of NHE2, while the 75 kDa protein is an unglycosylated form. Thus, unlike NHE1, which is N- and O-glycosylated, NHE2 has only O-linked glycosylation.

Original languageEnglish (US)
Pages (from-to)12954-12961
Number of pages8
JournalBiochemistry®
Volume33
Issue number44
StatePublished - 1994

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Sialoglycoproteins
Sodium-Hydrogen Antiporter
Glycosylation
Proteins
Glycoside Hydrolases
Neuraminidase
Membranes
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Antibodies
Protein S
Glutathione Transferase
Cell membranes
Digestion
Protein Isoforms
Coloring Agents
Western Blotting
Cell Membrane
Fusion reactions
Staining and Labeling
Rabbits

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tse, C. M., Levine, S. A., Yun, C. H. C., Khurana, S., & Donowitz, M. (1994). Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein. Biochemistry®, 33(44), 12954-12961.

Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein. / Tse, Chung Ming; Levine, Susan A.; Yun, C. H Chris; Khurana, Seema; Donowitz, Mark.

In: Biochemistry®, Vol. 33, No. 44, 1994, p. 12954-12961.

Research output: Contribution to journalArticle

Tse, CM, Levine, SA, Yun, CHC, Khurana, S & Donowitz, M 1994, 'Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein', Biochemistry®, vol. 33, no. 44, pp. 12954-12961.
Tse, Chung Ming ; Levine, Susan A. ; Yun, C. H Chris ; Khurana, Seema ; Donowitz, Mark. / Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein. In: Biochemistry®. 1994 ; Vol. 33, No. 44. pp. 12954-12961.
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abstract = "A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-α-D-galactosaminide (BzαGalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the BzαGalNAc treatment of PS120/NHE2 cells. These results suggest that the 85 kDa protein is an O-glycosylated form of NHE2, while the 75 kDa protein is an unglycosylated form. Thus, unlike NHE1, which is N- and O-glycosylated, NHE2 has only O-linked glycosylation.",
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N2 - A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-α-D-galactosaminide (BzαGalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the BzαGalNAc treatment of PS120/NHE2 cells. These results suggest that the 85 kDa protein is an O-glycosylated form of NHE2, while the 75 kDa protein is an unglycosylated form. Thus, unlike NHE1, which is N- and O-glycosylated, NHE2 has only O-linked glycosylation.

AB - A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-α-D-galactosaminide (BzαGalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the BzαGalNAc treatment of PS120/NHE2 cells. These results suggest that the 85 kDa protein is an O-glycosylated form of NHE2, while the 75 kDa protein is an unglycosylated form. Thus, unlike NHE1, which is N- and O-glycosylated, NHE2 has only O-linked glycosylation.

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