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 language | English (US) |
|---|---|
| Pages (from-to) | 12954-12961 |
| Number of pages | 8 |
| Journal | Biochemistry® |
| Volume | 33 |
| Issue number | 44 |
| State | Published - 1994 |
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Cite this
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 journal › Article
}
TY - JOUR
T1 - Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein
AU - Tse, Chung Ming
AU - Levine, Susan A.
AU - Yun, C. H Chris
AU - Khurana, Seema
AU - Donowitz, Mark
PY - 1994
Y1 - 1994
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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UR - http://www.scopus.com/inward/citedby.url?scp=0028030205&partnerID=8YFLogxK
M3 - Article
C2 - 7524659
AN - SCOPUS:0028030205
VL - 33
SP - 12954
EP - 12961
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 44
ER -