Role of Glycosylation in the Organic Anion Transporter OAT1

Abstract

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519–1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential function. In a replacement of asparagines in both mOAT1 and hOAT1 the of the transporters to the In we the that Asp-39 is involved in substrate of glycosylation at individual sites is not and glycosylation an important role in the of the is the and of glycosylation of and important the of the organic anion transporter Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519–1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential function. In a replacement of asparagines in both mOAT1 and hOAT1 the of the transporters to the In we the that Asp-39 is involved in substrate of glycosylation at individual sites is not and glycosylation an important role in the of the is the and of glycosylation of and important the of the organic anion transporter Organic anion transporters (OAT) organic anion organic mouse human organic anion organic mouse human play essential roles in the body disposition of clinically important anionic including antitumor drugs, antibiotics, antihypertensives, and G. by and G. and in the and In the transporters a transport to organic the the cells the this transport an to a an that is by a anion to the substrate the of transport to and the the of a substrate both its and the of the is in the is present in the and transport and to a putative and a and of to play a of roles including of of and of We previously G. J. Biol. Chem. that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting that glycosylation is essential mOAT1 function. this the on the and the roles of glycosylation in is not In the present study, we investigated the of of the sites in mOAT1 on its function and hOAT1, the human of is the human the glycosylation of hOAT1 was acid was and other transporters by of asparagine to glutamine of and and a acid at the of mOAT1 and G. J. Biol. Chem. G. J. Biol. Chem. showed that the the of the mutant by the in HeLa cells at and in HeLa cells and the G. cells at and transport and cell surface was of and and the in the the was by the and the well cells in in and was by the of in of by surface of hOAT1, and their the transporters in HeLa cells in as the was and the cells of on and the of the well of cells was of in in on was well was of the on to complete of the cells on in of inhibitor cells by at at of to the to the cell mOAT1 and hOAT1 in the of surface by and an of cell surface in and at and in and of at and on and in and at and to and by of HeLa cells in at in in and in cells cells at in and in the at cells and by to and the in on a of mOAT1 and and previously G. of that glycosylated in We showed that both mOAT1 and hOAT1 glycosylated in HeLa cells. of HeLa cells expressing mOAT1 and hOAT1 a an of an the the to a of the mOAT1 and showed that both transporters glycosylated in HeLa of the of Single of on the amino acid of and their sites in the as in sites at and hOAT1, sites at and of sites glycosylation, we the glycosylation by asparagine glutamine of cells expressing mOAT1 showed that in the of and had that of glycosylated but that of had a that was the as that of mutant their to the as that of suggesting that in mOAT1 and Asp-39 is not of cells expressing hOAT1 showed that in the of and had that of glycosylated but that of had a the as that of mutant their to the as that of hOAT1, suggesting that in hOAT1, and is not of of the putative glycosylation sites as of the effect of of putative glycosylation transporters by asparagine glutamine at glycosylation site of mOAT1 and hOAT1 and in HeLa cells. on surface as without by on and of the of Single of the of disrupting putative glycosylation sites by the of substrate in cells. in the replacement of Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 Asp-39 is not glycosylated in mOAT1 but is glycosylated in hOAT1 the loss of transport activity of in both mOAT1 and hOAT1 not because of deglycosylation of Asp-39 per se but rather because of the change in amino acid the cell surface of is that of both mOAT1 and hOAT1 the loss of function of this mutant is not because of the of the transporters to the but is rather because of the substrate asparagines at other sites had no effect on transport activity both mOAT1 and hOAT1 suggesting that glycosylation at individual sites is not the transport function of both of the effect of of putative glycosylation transporters by asparagine glutamine at glycosylation site of mOAT1 and hOAT1 and in HeLa cells by as the of the the of the of of investigated the effect of of of the glycosylation In replacement of and an without is the as that of indicating that no sites is important to that of the glycosylation sites the of the mOAT1 was as that of mOAT1 the of the mutant was to that of not suggesting that glycosylation is the of the transporter the In hOAT1, and was not glycosylation this is that the mutant a of the of hOAT1, and to a as in in the of a of of the to the of that an glycosylation site the glycosylation sites the that is the glycosylation we mutant this mutant a the of the hOAT1, both in the and of that glycosylated other sites is important to that hOAT1 to the as the of glycosylation the of of the and the was to that of the not the of the mutant was of the hOAT1, and the of the mutant was of the effect of of putative glycosylation transporters by the asparagines glutamine in mOAT1 and hOAT1 and in HeLa cells. cells. without by on and of the of of of this of to to the was by the was as in cells mOAT1 and hOAT1, in the the mOAT1 and the hOAT1 showed that the cells to the of that glycosylation is of both mOAT1 and hOAT1 to the of the effect of deglycosylation of mOAT1 and hOAT1 on their hOAT1, and their in HeLa cells. cells and as showed that cells to the of the of of of of glycosylation sites on the function of mOAT1 and hOAT1 no individual glycosylation site is mOAT1 of the glycosylation sites resulted in a in transport activity hOAT1 showed no transport activity of the effect of of putative glycosylation transporters by the asparagines glutamine in mOAT1 and hOAT1 in HeLa cells by as the of the the is to function as an G. organic anion the cells to organic anion of the cells. deglycosylation of mOAT1 cells expressing the mutant by to the without substrate in of was both mOAT1 and its mutant cells to an substrate as without mutant was to the by an inhibitor to that of mOAT1 that the mOAT1 the of the of of expressing mOAT1 and its mutant by an to by mOAT1 and its mutant in HeLa by in the of play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. of the is the of sites glycosylation in the the of of sites is by that the of mOAT1 in mouse was the was to the of the G. of the in the of mOAT1 by in is by G. J. Biol. Chem. and the of COS-7 cells resulted in an complete loss of organic anion the that of the in the in cells G. J. Biol. Chem. In that mOAT1 is a and glycosylation an important role mOAT1 the present we to the glycosylation of mOAT1 and the role of hOAT1, the human of is the human the glycosylation of hOAT1 was on the both mOAT1 and hOAT1 asparagines in the to the sites of sites is glycosylation, we by asparagine glutamine in of that of the sites glycosylation both mOAT1 and at sites the of the and in hOAT1 is not glycosylated other glycosylation sites glycosylation the at other sites to the of glycosylation at in of the this asparagine an substrate the in the of the effect of a of the glycosylation sites that disrupting Asp-39 in both mOAT1 and hOAT1 resulted in an complete loss of transport activity without affecting the cell surface of Asp-39 in mOAT1 is not glycosylation, Asp-39 in hOAT1 is glycosylated. that the loss of function is not because of deglycosylation of Asp-39 per se but is rather because of the change of an amino acid in an important in the Asp-39 in mOAT1 is in the as Asp-39 in hOAT1, amino acid the of the is likely that the of the the Single replacement of asparagines at other sites had no effect on transport activity mOAT1 hOAT1, suggesting that glycosylation at individual sites is not essential function. In the glycosylation sites the of mOAT1 and hOAT1 was that of the mOAT1 and hOAT1 in the that glycosylation an essential role the of transporters the is important to that the loss of the surface in because of the in amino acid and not because of the deglycosylation per G. J. Biol. Chem. in COS-7 cells tunicamycin, a in the asparagines in the transporter not that deglycosylation is to the of the transporters the the present that Asp-39 in both mOAT1 and hOAT1 an important role in the substrate of glycosylation at individual sites is not the transport and glycosylation is essential the of the transporter to the is the and of glycosylation in and important the of the organic transporter Organic anion transporters (OAT) organic anion organic mouse human organic anion organic mouse human play essential roles in the body disposition of clinically important anionic including antitumor drugs, antibiotics, antihypertensives, and G. by and G. and in the and In the transporters a transport to organic the the cells the this transport an to a an that is by a anion to the substrate the of transport to and the the of a substrate both its and the of the is in the is present in the and transport and to a putative and a and of to play a of roles including of of and of We previously G. J. Biol. Chem. that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting that glycosylation is essential mOAT1 function. this the on the and the roles of glycosylation in is not In the present study, we investigated the of of the sites in mOAT1 on its function and hOAT1, the human of is the human the glycosylation of hOAT1 was acid was and other transporters by of asparagine to glutamine of and and a acid at the of mOAT1 and G. J. Biol. Chem. G. J. Biol. Chem. showed that the the of the mutant by the in HeLa cells at and in HeLa cells and the G. cells at and transport and cell surface was of and and the in the the was by the and the well cells in in and was by the of in of by surface of hOAT1, and their the transporters in HeLa cells in as the was and the cells of on and the of the well of cells was of in in on was well was of the on to complete of the cells on in of inhibitor cells by at at of to the to the cell mOAT1 and hOAT1 in the of surface by and an of cell surface in and at and in and of at and on and in and at and to and by of HeLa cells in at in in and in cells cells at in and in the at cells and by to and the in on a acid was and other acid was and other transporters by of asparagine to glutamine of and and a acid at the of mOAT1 and G. J. Biol. Chem. G. J. Biol. Chem. showed that the the of the mutant by the in HeLa cells at and in HeLa cells and the G. cells at and transport and cell surface was of and and the in the the was by the and the well cells in in and was by the of in of by surface of hOAT1, and their the transporters in HeLa cells in as the was and the cells of on and the of the well of cells was of in in on was well was of the on to complete of the cells on in of inhibitor cells by at at of to the to the cell mOAT1 and hOAT1 in the of surface by and an of cell surface in and at and in and of at and on and in and at and to and by of HeLa cells in at in in and in cells cells at in and in the at cells and by to and the in on a transporters by of asparagine to glutamine of and and a acid at the of mOAT1 and G. J. Biol. Chem. G. J. Biol. Chem. showed that the the of the mutant by the in HeLa cells at and in HeLa cells and the G. cells at and transport and cell surface was of and and the in the the was by the and the well cells in in and was by the of in of by surface of hOAT1, and their the transporters in HeLa cells in as the was and the cells of on and the of the well of cells was of in in on was well was of the on to complete of the cells on in of inhibitor cells by at at of to the to the cell mOAT1 and hOAT1 in the of surface by and an of cell surface in and at and in and of at and on and in and at and to and by of HeLa cells in at in in and in cells cells at in and in the at cells and by to and the in on a of mOAT1 and and previously G. of that glycosylated in We showed that both mOAT1 and hOAT1 glycosylated in HeLa cells. of HeLa cells expressing mOAT1 and hOAT1 a an of an the the to a of the mOAT1 and showed that both transporters glycosylated in HeLa of the of Single of on the amino acid of and their sites in the as in sites at and hOAT1, sites at and of sites glycosylation, we the glycosylation by asparagine glutamine of cells expressing mOAT1 showed that in the of and had that of glycosylated but that of had a that was the as that of mutant their to the as that of suggesting that in mOAT1 and Asp-39 is not of cells expressing hOAT1 showed that in the of and had that of glycosylated but that of had a the as that of mutant their to the as that of hOAT1, suggesting that in hOAT1, and is not of the effect of of putative glycosylation transporters by asparagine glutamine at glycosylation site of mOAT1 and hOAT1 and in HeLa cells. on surface as without by on and of the of Single of the of disrupting putative glycosylation sites by the of substrate in cells. in the replacement of Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 Asp-39 is not glycosylated in mOAT1 but is glycosylated in hOAT1 the loss of transport activity of in both mOAT1 and hOAT1 not because of deglycosylation of Asp-39 per se but rather because of the change in amino acid the cell surface of is that of both mOAT1 and hOAT1 the loss of function of this mutant is not because of the of the transporters to the but is rather because of the substrate asparagines at other sites had no effect on transport activity both mOAT1 and hOAT1 suggesting that glycosylation at individual sites is not the transport function of both of the effect of of putative glycosylation transporters by asparagine glutamine at glycosylation site of mOAT1 and hOAT1 and in HeLa cells by as the of the the of the of of investigated the effect of of of the glycosylation In replacement of and an without is the as that of indicating that no sites is important to that of the glycosylation sites the of the mOAT1 was as that of mOAT1 the of the mutant was to that of not suggesting that glycosylation is the of the transporter the In hOAT1, and was not glycosylation this is that the mutant a of the of hOAT1, and to a as in in the of a of of the to the of that an glycosylation site the glycosylation sites the that is the glycosylation we mutant this mutant a the of the hOAT1, both in the and of that glycosylated other sites is important to that hOAT1 to the as the of glycosylation the of of the and the was to that of the not the of the mutant was of the hOAT1, and the of the mutant was of the effect of of putative glycosylation transporters by the asparagines glutamine in mOAT1 and hOAT1 and in HeLa cells. cells. without by on and of the of of of this of to to the was by the was as in cells mOAT1 and hOAT1, in the the mOAT1 and the hOAT1 showed that the cells to the of that glycosylation is of both mOAT1 and hOAT1 to the of the effect of deglycosylation of mOAT1 and hOAT1 on their hOAT1, and their in HeLa cells. cells and as showed that cells to the of the of of of of glycosylation sites on the function of mOAT1 and hOAT1 no individual glycosylation site is mOAT1 of the glycosylation sites resulted in a in transport activity hOAT1 showed no transport activity of the effect of of putative glycosylation transporters by the asparagines glutamine in mOAT1 and hOAT1 in HeLa cells by as the of the the is to function as an G. organic anion the cells to organic anion of the cells. deglycosylation of mOAT1 cells expressing the mutant by to the without substrate in of was both mOAT1 and its mutant cells to an substrate as without mutant was to the by an inhibitor to that of mOAT1 that the mOAT1 the of the of of expressing mOAT1 and its mutant by an to by mOAT1 and its mutant in HeLa by in the of of mOAT1 and and previously G. of that glycosylated in We showed that both mOAT1 and hOAT1 glycosylated in HeLa cells. of HeLa cells expressing mOAT1 and hOAT1 a an of an the the to a of the mOAT1 and showed that both transporters glycosylated in HeLa cells. of the of Single of on the amino acid of and their sites in the as in sites at and hOAT1, sites at and of sites glycosylation, we the glycosylation by asparagine glutamine of cells expressing mOAT1 showed that in the of and had that of glycosylated but that of had a that was the as that of mutant their to the as that of suggesting that in mOAT1 and Asp-39 is not of cells expressing hOAT1 showed that in the of and had that of glycosylated but that of had a the as that of mutant their to the as that of hOAT1, suggesting that in hOAT1, and is not of the of Single of the of disrupting putative glycosylation sites by the of substrate in cells. in the replacement of Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 Asp-39 is not glycosylated in mOAT1 but is glycosylated in hOAT1 the loss of transport activity of in both mOAT1 and hOAT1 not because of deglycosylation of Asp-39 per se but rather because of the change in amino acid the cell surface of is that of both mOAT1 and hOAT1 the loss of function of this mutant is not because of the of the transporters to the but is rather because of the substrate asparagines at other sites had no effect on transport activity both mOAT1 and hOAT1 suggesting that glycosylation at individual sites is not the transport function of both of the of of investigated the effect of of of the glycosylation In replacement of and an without is the as that of indicating that no sites is important to that of the glycosylation sites the of the mOAT1 was as that of mOAT1 the of the mutant was to that of not suggesting that glycosylation is the of the transporter the In hOAT1, and was not glycosylation this is that the mutant a of the of hOAT1, and to a as in in the of a of of the to the of that an glycosylation site the glycosylation sites the that is the glycosylation we mutant this mutant a the of the hOAT1, both in the and of that glycosylated other sites is important to that hOAT1 to the as the of glycosylation the of of the and the was to that of the not the of the mutant was of the hOAT1, and the of the mutant was of the of of of this of to to the was by the was as in cells mOAT1 and hOAT1, in the the mOAT1 and the hOAT1 showed that the cells to the of that glycosylation is of both mOAT1 and hOAT1 to the of the of of of of glycosylation sites on the function of mOAT1 and hOAT1 no individual glycosylation site is mOAT1 of the glycosylation sites resulted in a in transport activity hOAT1 showed no transport activity is to function as an G. organic anion the cells to organic anion of the cells. deglycosylation of mOAT1 cells expressing the mutant by to the without substrate in of was both mOAT1 and its mutant cells to an substrate as without mutant was to the by an inhibitor to that of mOAT1 that the mOAT1 the of the play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. of the is the of sites glycosylation in the the of of sites is by that the of mOAT1 in mouse was the was to the of the G. of the in the of mOAT1 by in is by G. J. Biol. Chem. and the of COS-7 cells resulted in an complete loss of organic anion the that of the in the in cells G. J. Biol. Chem. In that mOAT1 is a and glycosylation an important role mOAT1 the present we to the glycosylation of mOAT1 and the role of hOAT1, the human of is the human the glycosylation of hOAT1 was on the both mOAT1 and hOAT1 asparagines in the to the sites of sites is glycosylation, we by asparagine glutamine in of that of the sites glycosylation both mOAT1 and at sites the of the and in hOAT1 is not glycosylated other glycosylation sites glycosylation the at other sites to the of glycosylation at in of the this asparagine an substrate the in the of the effect of a of the glycosylation sites that disrupting Asp-39 in both mOAT1 and hOAT1 resulted in an complete loss of transport activity without affecting the cell surface of Asp-39 in mOAT1 is not glycosylation, Asp-39 in hOAT1 is glycosylated. that the loss of function is not because of deglycosylation of Asp-39 per se but is rather because of the change of an amino acid in an important in the Asp-39 in mOAT1 is in the as Asp-39 in hOAT1, amino acid the of the is likely that the of the the Single replacement of asparagines at other sites had no effect on transport activity mOAT1 hOAT1, suggesting that glycosylation at individual sites is not essential function. In the glycosylation sites the of mOAT1 and hOAT1 was that of the mOAT1 and hOAT1 in the that glycosylation an essential role the of transporters the is important to that the loss of the surface in because of the in amino acid and not because of the deglycosylation per G. J. Biol. Chem. in COS-7 cells tunicamycin, a in the asparagines in the transporter not that deglycosylation is to the of the transporters the the present that Asp-39 in both mOAT1 and hOAT1 an important role in the substrate of glycosylation at individual sites is not the transport and glycosylation is essential the of the transporter to the is the and of glycosylation in and important the of the organic transporter play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. of the is the of sites glycosylation in the the of of sites is by that the of mOAT1 in mouse was the was to the of the G. of the in the of mOAT1 by in is by G. J. Biol. Chem. and the of COS-7 cells resulted in an complete loss of organic anion the that of the in the in cells G. J. Biol. Chem. In that mOAT1 is a and glycosylation an important role mOAT1 function. In the present we to the glycosylation of mOAT1 and the role of hOAT1, the human of is the human the glycosylation of hOAT1 was on the both mOAT1 and hOAT1 asparagines in the to the sites of sites is glycosylation, we by asparagine glutamine in of that of the sites glycosylation both mOAT1 and at sites the of the and in hOAT1 is not glycosylated other glycosylation sites glycosylation the at other sites to the of glycosylation at in of the this asparagine an substrate the in the of the effect of a of the glycosylation sites that disrupting Asp-39 in both mOAT1 and hOAT1 resulted in an complete loss of transport activity without affecting the cell surface of Asp-39 in mOAT1 is not glycosylation, Asp-39 in hOAT1 is glycosylated. that the loss of function is not because of deglycosylation of Asp-39 per se but is rather because of the change of an amino acid in an important in the Asp-39 in mOAT1 is in the as Asp-39 in hOAT1, amino acid the of the is likely that the of the the Single replacement of asparagines at other sites had no effect on transport activity mOAT1 hOAT1, suggesting that glycosylation at individual sites is not essential function. In the glycosylation sites the of mOAT1 and hOAT1 was that of the mOAT1 and hOAT1 in the that glycosylation an essential role the of transporters the is important to that the loss of the surface in because of the in amino acid and not because of the deglycosylation per G. J. Biol. Chem. in COS-7 cells tunicamycin, a in the asparagines in the transporter not that deglycosylation is to the of the transporters the In the present that Asp-39 in both mOAT1 and hOAT1 an important role in the substrate of glycosylation at individual sites is not the transport and glycosylation is essential the of the transporter to the is the and of glycosylation in and important the of the organic transporter