642nd Meeting Southampton Held a t the University of Southampton 7- I0 April I992
Biochemical SocietyTransactions ( 1 992) 20 Examination of the glycosidation state of five members of the human facilitative glucose transporter family.
a GLUT 4 -200
ALISON M. BRANT, E. MICHAEL GIBBS' and GWYN W. COULD.
-116.5
Department of Biochemistry, University of Glasgow, Glasgow G I 2 8 0 , Scotland. and *Pfizer Central Research, Groton, CT. 06340, U.S.A. Glucose transport a c m s the plasma membrane of animal cells is mediated by a family of highly homologous proteins. These transporters are the products of distinct genes and are expressed in a highly regulated and co-ordinated fashion [l]. Computer analysis of the primary sequence of all five family members suggests that the transporter proteins all possess twelve putative transmembrane helices, have intracellularly loca!ised N- and Ctermini and contain large hydrophillic domains located between helices 1 and 2 (extracellular) and helices 6 and 7 (intracellular) [2]. Such analysis has further revealed that the five transporters share between 40 and 58% identity at the amino acid level, the most extensive identity being found within the putative transmembrane sections of the protein. This observation is probably consistant with the proposal that the glucose molecule is moved across the membrane via a water-lined 'pore' generated by arrangement of the transmembrane helices [3]. Sequence identity within the hydrophillic N- and Cterminal domains is minimal (hence providing excellent targets for anti-peptide antibodies) but within the extracellularloop of GLUTs 1,3,4 and 5 a consensus sequence for N-linked glycosidation is present (Table I). Previous studies have demonstrated that the erythrocytetype glucose transporter (GLUT 1) is a glycoprotein with a heterogeneous carbohydrate moeity incorporated via N-linked glycosidation to Asn45, located in this predicted extracellularloop. The demonstration that GLUT 1 is a glycoprotein may be readily achieved by the digestion of erythrocyte membranes with Endoglycosidase F and comparison of the relative mobility of the
Table I Amino Acid Sequence o f ' the Extracellular Loops of t h e Human Glucose Transporter Family. GLUT GLUT GLUT GLUT GLUT
1 .NAPQKVIEEFYNQTWVHRYGESILPTT.. 2 .NAPQQVIISHYRHVLGVPLDDRKAINNYV 3 .NAPEKIIKIFINKTLTDKGNAPPSEVL.. 4 .NAPQKVIEQSYNETWLGRQGPEGPSSIPP 5 .NSPALLMQQFYNETYYGRTQEFMEDFP..
The sequences of the extracellular domains of GLUTs 1 to 5 are presented, written from left (N-terminal) to right (C-terminal). The consensus sequence for N-linked glycosidation is in bold type. GLUT 1 protein upon SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-GLUT 1 antibodies. Prior to digestion the protein migrates as a broad band of Mr about 5055 kDa; upon Endoglycosidase F treatment the immunoreactive species migrates as a sharper band of Mr 46 kDa [4]. We have used a similar approach to evaluate the glycosidation state of GLUTs 2,3,4 and 5, using the anti-peptide antibodies described [5]. Rat or mouse liver plasma membranes (GLUT 2), mouse brain membranes (GLUT 3), 3T3-Ll adipocyte membranes (GLUT 1 and GLUT 4) and human duodenal membranes (GLUT 5) were prepared as described r61. Aliauots of membranes (100150 pg>-wekresuspended in 56 mM potassium phosphate, pH 5.0, 20 mM EDTA, 2% Triton X-100, 0.2% SDS. 1% 2mercaptoethanol and incubated for 24 h with 0.35 U Nglycosidase F and 5 pg neuraminidase at 37'. Samples were briefly vortexed during the incubation. Following incubation, the proteins were precipitated using trichloroacetic acid, resuspended in SDS-PAGE sample buffer, electrophoresed and immunoblotted with the appropriate antibodies. Using this approach, we have demonstrated that the murine homologues of GLUT 4 (Figl), GLUT 3 (Fig.2) and GLUT 1 (data not shown) are indeed glycosylated, since in all
235s
-80 -49
"r
1
I5
d
2
3
Figure 1.
1
2 Figure 2.
Figure 1. Effect of deglycosidation on the mobility of GLUT 4. 50 wg of 3 n - L l adipocyte membrane protein was incubated overnight at 37" with (lane 3) or without (lane 2) neurdminidaseand endoglycosidaseF as described. Samples containingequal amounts of were eleceophoresedand immunoblottedwith antibodies against the C-terminal 14 amino acids of GLUT 4. Lane 1 contains 50 wg of membranes without overnight treatement. Figure 2. Effect of deglycosidation on the mobility of GLUT 3. 115 Kg of mouse brain membranes were incubated in the presence (lane 2) or absence (lane 1) of neuminidase and endoglycosidaseF as described. Samples containing equal amounts (10 wg) of membrane protein were then elecwphoresed and immunoblotted with antibodies against the C-terminal14 amino acids of the m@ne isoform of GLUT 3.
cases after glycosidase treatment the immunoreactive species exhibited a profound decrease in Mr on SDS-PAGE following electrophoresis. GLUT 3 in the native state migrated as a band at 49 kDa. Upon digestion, the apparant molecular weight decresed to 43 m a . Similarly, GLUT 4 before digestion migrated as a broad band at 45 kDa which decreased to a sharper band at 43 kDa following digestion. In a series of experiments not shown here, we have also demonstrated that GLUT 5 in human small intestine migrates as a broad band of about 55 kDa which decreased upon endoglycosidase F digestion to a sharp band at 50 kDa (Peter R. Shepherd, E.M.G., G.W.G., and Barbara B. Kahn, manuscript submitted). We have attempted similar experiments with both rat and mouse liver plasma membranes to evaluate the glycosidation state of GLUT 2. In rat liver, GLUT 2 migrates as a series of bands at Mr 56 (broad band, possibly a dimer), 42 and 31 kDa. We have examined the effects of both Endogycosidase F/neuraminidase,Nglycanase and also neuraminidase/O-glycanase on the relative mobility of these species. We have been unable to demonstrate any shift in mobility following these treatments in several different preparations of rat or mouse liver plasma membranes (data not shown). Control experiments have demonstrated that the low level of GLUT 1 expressed in these membranes does effectively deglycosidate under these conditions. It is notable that GLUT 2 is the only transporter isofom identified to date which does not contain a consensus sequence for glycosidation in the extracellular loop (Table I). These results indicate that GLUT 3, 4 and 5 are, like GLUT 1, N-linked glycoproteins. It is likely that this glycosylation is via the conserved Asn residue in the extracellular loop between transmembrane helices 1 and 2. We have been unable to demonstrate any effect of N- or 0-glycanases on GLUT 2 mobility. This suggests that the different species seen upon SDSPAGE and immunoblotting may represent a distinct posttranslational modification in this isoform. [ll Gould, G.W. & Bell, G.I.(1990) Trends Biochem. Sci. 15, 18-23. [21 Meuck1er.M..et al..(1985) Science 229,941-945. [31 Alvarez, J., et a1.,(1987)J. Biol. Chem. 262. 3502-3509. [41 Lienhard. G.E., et al.. (1983) Structure. and Fucntion of Membrane Proteins. 325-333. @ds: Quagliarello & Palmeri. Elsevier). [51 Brant, A.M., et al., Biochem. Soc.Trans,Ibid. [61 Gould, G.W., et al (1992) Diaktelogia, in press.
Biochemical SocietyTransactions ( 1 992) 20 Examination of the glycosidation state of five members of the human facilitative glucose transporter family.
a GLUT 4 -200
ALISON M. BRANT, E. MICHAEL GIBBS' and GWYN W. COULD.
-116.5
Department of Biochemistry, University of Glasgow, Glasgow G I 2 8 0 , Scotland. and *Pfizer Central Research, Groton, CT. 06340, U.S.A. Glucose transport a c m s the plasma membrane of animal cells is mediated by a family of highly homologous proteins. These transporters are the products of distinct genes and are expressed in a highly regulated and co-ordinated fashion [l]. Computer analysis of the primary sequence of all five family members suggests that the transporter proteins all possess twelve putative transmembrane helices, have intracellularly loca!ised N- and Ctermini and contain large hydrophillic domains located between helices 1 and 2 (extracellular) and helices 6 and 7 (intracellular) [2]. Such analysis has further revealed that the five transporters share between 40 and 58% identity at the amino acid level, the most extensive identity being found within the putative transmembrane sections of the protein. This observation is probably consistant with the proposal that the glucose molecule is moved across the membrane via a water-lined 'pore' generated by arrangement of the transmembrane helices [3]. Sequence identity within the hydrophillic N- and Cterminal domains is minimal (hence providing excellent targets for anti-peptide antibodies) but within the extracellularloop of GLUTs 1,3,4 and 5 a consensus sequence for N-linked glycosidation is present (Table I). Previous studies have demonstrated that the erythrocytetype glucose transporter (GLUT 1) is a glycoprotein with a heterogeneous carbohydrate moeity incorporated via N-linked glycosidation to Asn45, located in this predicted extracellularloop. The demonstration that GLUT 1 is a glycoprotein may be readily achieved by the digestion of erythrocyte membranes with Endoglycosidase F and comparison of the relative mobility of the
Table I Amino Acid Sequence o f ' the Extracellular Loops of t h e Human Glucose Transporter Family. GLUT GLUT GLUT GLUT GLUT
1 .NAPQKVIEEFYNQTWVHRYGESILPTT.. 2 .NAPQQVIISHYRHVLGVPLDDRKAINNYV 3 .NAPEKIIKIFINKTLTDKGNAPPSEVL.. 4 .NAPQKVIEQSYNETWLGRQGPEGPSSIPP 5 .NSPALLMQQFYNETYYGRTQEFMEDFP..
The sequences of the extracellular domains of GLUTs 1 to 5 are presented, written from left (N-terminal) to right (C-terminal). The consensus sequence for N-linked glycosidation is in bold type. GLUT 1 protein upon SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-GLUT 1 antibodies. Prior to digestion the protein migrates as a broad band of Mr about 5055 kDa; upon Endoglycosidase F treatment the immunoreactive species migrates as a sharper band of Mr 46 kDa [4]. We have used a similar approach to evaluate the glycosidation state of GLUTs 2,3,4 and 5, using the anti-peptide antibodies described [5]. Rat or mouse liver plasma membranes (GLUT 2), mouse brain membranes (GLUT 3), 3T3-Ll adipocyte membranes (GLUT 1 and GLUT 4) and human duodenal membranes (GLUT 5) were prepared as described r61. Aliauots of membranes (100150 pg>-wekresuspended in 56 mM potassium phosphate, pH 5.0, 20 mM EDTA, 2% Triton X-100, 0.2% SDS. 1% 2mercaptoethanol and incubated for 24 h with 0.35 U Nglycosidase F and 5 pg neuraminidase at 37'. Samples were briefly vortexed during the incubation. Following incubation, the proteins were precipitated using trichloroacetic acid, resuspended in SDS-PAGE sample buffer, electrophoresed and immunoblotted with the appropriate antibodies. Using this approach, we have demonstrated that the murine homologues of GLUT 4 (Figl), GLUT 3 (Fig.2) and GLUT 1 (data not shown) are indeed glycosylated, since in all
235s
-80 -49
"r
1
I5
d
2
3
Figure 1.
1
2 Figure 2.
Figure 1. Effect of deglycosidation on the mobility of GLUT 4. 50 wg of 3 n - L l adipocyte membrane protein was incubated overnight at 37" with (lane 3) or without (lane 2) neurdminidaseand endoglycosidaseF as described. Samples containingequal amounts of were eleceophoresedand immunoblottedwith antibodies against the C-terminal 14 amino acids of GLUT 4. Lane 1 contains 50 wg of membranes without overnight treatement. Figure 2. Effect of deglycosidation on the mobility of GLUT 3. 115 Kg of mouse brain membranes were incubated in the presence (lane 2) or absence (lane 1) of neuminidase and endoglycosidaseF as described. Samples containing equal amounts (10 wg) of membrane protein were then elecwphoresed and immunoblotted with antibodies against the C-terminal14 amino acids of the m@ne isoform of GLUT 3.
cases after glycosidase treatment the immunoreactive species exhibited a profound decrease in Mr on SDS-PAGE following electrophoresis. GLUT 3 in the native state migrated as a band at 49 kDa. Upon digestion, the apparant molecular weight decresed to 43 m a . Similarly, GLUT 4 before digestion migrated as a broad band at 45 kDa which decreased to a sharper band at 43 kDa following digestion. In a series of experiments not shown here, we have also demonstrated that GLUT 5 in human small intestine migrates as a broad band of about 55 kDa which decreased upon endoglycosidase F digestion to a sharp band at 50 kDa (Peter R. Shepherd, E.M.G., G.W.G., and Barbara B. Kahn, manuscript submitted). We have attempted similar experiments with both rat and mouse liver plasma membranes to evaluate the glycosidation state of GLUT 2. In rat liver, GLUT 2 migrates as a series of bands at Mr 56 (broad band, possibly a dimer), 42 and 31 kDa. We have examined the effects of both Endogycosidase F/neuraminidase,Nglycanase and also neuraminidase/O-glycanase on the relative mobility of these species. We have been unable to demonstrate any shift in mobility following these treatments in several different preparations of rat or mouse liver plasma membranes (data not shown). Control experiments have demonstrated that the low level of GLUT 1 expressed in these membranes does effectively deglycosidate under these conditions. It is notable that GLUT 2 is the only transporter isofom identified to date which does not contain a consensus sequence for glycosidation in the extracellular loop (Table I). These results indicate that GLUT 3, 4 and 5 are, like GLUT 1, N-linked glycoproteins. It is likely that this glycosylation is via the conserved Asn residue in the extracellular loop between transmembrane helices 1 and 2. We have been unable to demonstrate any effect of N- or 0-glycanases on GLUT 2 mobility. This suggests that the different species seen upon SDSPAGE and immunoblotting may represent a distinct posttranslational modification in this isoform. [ll Gould, G.W. & Bell, G.I.(1990) Trends Biochem. Sci. 15, 18-23. [21 Meuck1er.M..et al..(1985) Science 229,941-945. [31 Alvarez, J., et a1.,(1987)J. Biol. Chem. 262. 3502-3509. [41 Lienhard. G.E., et al.. (1983) Structure. and Fucntion of Membrane Proteins. 325-333. @ds: Quagliarello & Palmeri. Elsevier). [51 Brant, A.M., et al., Biochem. Soc.Trans,Ibid. [61 Gould, G.W., et al (1992) Diaktelogia, in press.
