Purification and Characterization of Mouse Soluble Receptor for Advanced Glycation End Products (sRAGE)

Abstract

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimer's disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimer's disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans. The receptor for advanced glycation end products (RAGE) 1The abbreviations used are: RAGE, receptor for advanced glycation end products; sRAGE, soluble RAGE; RT-PCR, reverse transcription PCR; LC-MS/MS, liquid chromatography tandem mass spectrometry; MALDI-MS, matrix-assisted laser desorption-ionization mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; PNGase F, N-glycosidase F; ECM, extracellular matrix.1The abbreviations used are: RAGE, receptor for advanced glycation end products; sRAGE, soluble RAGE; RT-PCR, reverse transcription PCR; LC-MS/MS, liquid chromatography tandem mass spectrometry; MALDI-MS, matrix-assisted laser desorption-ionization mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; PNGase F, N-glycosidase F; ECM, extracellular matrix. is a member of the immunoglobulin superfamily of cell surface receptors (1Neeper M. Schmidt A.M. Brett J. Yan J. is of extracellular a and a that is for RAGE-mediated signaling J. of of which advanced glycation end and in A.M. Yan Yan in humans, a cell signaling is in in the of the transcription factor J. J. M. J. Schmidt A.M. M. M. J. M. M. M. M. M. Schmidt A.M. Schmidt A.M. J. the RAGE J. Schmidt A.M. J. that to a such that RAGE is its In RAGE and its This is to the that RAGE is in is of chronic Alzheimer's and J. M. J. Schmidt A.M. Schmidt A.M. M. Schmidt A.M. J. M. M. J. Schmidt A.M. J. has a secreted soluble RAGE or sRAGE. a is secreted and acts as a sRAGE has been in a number of cell and animal models of RAGE-mediated successfully or RAGE signaling such as and Schmidt A.M. J. Yan Schmidt A.M. J. J. Schmidt A.M. J. M. J. Schmidt A.M. of the Yan J. J. Schmidt A.M. M. and cell and M. Schmidt A.M. sRAGE has been used in has been to biochemically characterize mouse sRAGE and the mechanism of its and M. J. found that in humans, sRAGE is produced by alternative splicing of RAGE mRNA. The of the and RAGE and sRAGE to carboxyl-terminal data that that mouse sRAGE is produced by carboxyl-terminal alternative the glycosylation and disulfide of sRAGE and a for sRAGE from mouse that a of sRAGE that the extracellular of from mouse from used as in by and was to the to J. in to of a and the was as was to the and was a and a The was was a of from the was and to The was to a and proteins a of a of sRAGE by mouse as J. and in from chromatography to as and to a The was the for the for the and, a of and as and of the chromatography was a liquid chromatography by was as J. of of sRAGE was as J. by and to mouse RAGE that sRAGE, or was mouse as J. or a mouse the the mouse RAGE RAGE found in the number to which was as for and for of for for and for and a for was and conditions as for of for for and for and a for of sRAGE for of sRAGE was and in the and and in the was in to the and of or of or of for a mass to The was used to the and a The reverse a high The was a of and from in to in data the for of the a and the data and as a The used to the data the from sRAGE and was a mass in in or of mouse RAGE was by for of sRAGE was of The was by to of sRAGE, the by in from was by reverse-phase chromatography a from to of and used to the the from a was as of by a disulfide of was to to that the mass to the mass of the Finally, from was and by to whether that the by a disulfide of of sRAGE was for in and was by the of of PNGase and the was for of sRAGE in was of for of was and the was for sRAGE PNGase by and as by the of of sRAGE from was to and as J. of of the to the and was to and by PNGase from of sRAGE from used as for sRAGE sRAGE is found in mouse J. to was to by chromatography and the of mouse of sRAGE from mouse as The was to chromatography and of and in a and the was mouse of to determine the of the mouse sRAGE that the of sRAGE was of the the of the mouse RAGE of mouse RAGE found in the number the is the may and to the which is in determine whether is the the of mouse sRAGE, the was and to The was to This to by the the and was as the was of the sRAGE the is to in for mouse sRAGE. sRAGE was or and to for is the of to the the of the The of the a the The the which our data of of mouse a is mouse sRAGE was and the by was used to determine which the indicated of and was used to that the of the in of is by a disulfide The disulfide sRAGE is in of by a disulfide the sRAGE from M. J. that sRAGE is a of alternative splicing of RAGE mRNA. The alternative is of the and results in a that a is for sRAGE. RAGE and sRAGE carboxyl-terminal whether mouse sRAGE is produced by alternative to alternative RAGE used mouse or a mouse as a in or to end of the RAGE as as the to the in and in to a that alternative splicing to that in humans. found of alternative RAGE to mouse RAGE and indicated by to and sRAGE from a that found by that our to alternative mouse RAGE is to of our to RAGE used in to mouse RAGE and indicated in mouse RAGE found in the data number mouse RAGE found in the data number used to successfully mouse mouse RAGE found in the data number used to successfully mouse mouse RAGE found in the data number and indicated in mouse RAGE found in the data number used to successfully mouse in a the mouse sRAGE used mouse sRAGE was or and by to that and the data from the data from the from our of the and was by the the carboxyl-terminal end of mouse sRAGE from our data is to the which the sRAGE from that of RAGE as by M. J. determine whether the mass of sRAGE sRAGE was and to mouse sRAGE the in our its the of the found that mass of mouse sRAGE a the that mouse sRAGE is and a from mouse sRAGE by indicated of of of of sRAGE. in a of characterize mouse sRAGE, its disulfide was used for the that in from mouse sRAGE to from to to from to by a disulfide the of and by the to that the by a disulfide a of the of was to to that the mass to of of was and by to determine whether in that the of the sRAGE has and found that in disulfide a disulfide as and of of RAGE glycosylation was by J. found a in RAGE the same is true for sRAGE has the same as determine whether mouse sRAGE has and mouse sRAGE was PNGase F, or a of the of sRAGE by that mouse sRAGE is sRAGE was to was which the that mouse sRAGE is sRAGE and that of used sRAGE to and In addition, mass of mouse sRAGE and PNGase and a in of to and the of of the sRAGE that to of the by was by in and PNGase data used the to the of that has a glycosylation and has a or is sRAGE was PNGase and to to mouse J. mouse sRAGE that a to for and from used in the to determine the of the data for reported in reported in reported in of mass and by PNGase to for the of to from PNGase reported in mass and by PNGase to for the of to from PNGase in a a for sRAGE from mouse of which is in of sRAGE is in or J. This that sRAGE is to the extracellular matrix or cell and our that sRAGE has that sRAGE may to in the or cell in the or cell sRAGE may as a receptor by and from and cell surface RAGE and RAGE is to the that a in sRAGE that its to from the and the to has been that from the the M. J. Schmidt A.M. to determine whether the of is a of sRAGE of RAGE has been J. M. M. J. Schmidt A.M. The of in has to data that mouse sRAGE is produced by a mechanism from sRAGE, which is produced by alternative found for alternative RAGE in mouse or to a of RAGE, is likely that RAGE is to sRAGE. has been found to to sRAGE the J. in sRAGE from may that sRAGE of In to alternative sRAGE may by in a to that in mouse the the has been to in Yan M. Yan M. Schmidt A.M. J. sRAGE is in is likely that glycosylation is for the ability of sRAGE to bind its which in by J. a for sRAGE used a of glycosylation or by is to that is a the mass of mouse sRAGE as by and mass of sRAGE that is of sRAGE and sRAGE in is the of mouse sRAGE in and conditions, mouse sRAGE has been whether is in high mass or whether is sRAGE. our of sRAGE has been reported to Schmidt A.M. Yan Brett J. M. J. reported that RAGE to a results in a high mass by determine whether of in the mouse and is for the high mass in that sRAGE has which its in tissues. was found to that alternative splicing of RAGE to sRAGE in the mouse as was found for sRAGE. In the carboxyl-terminal of RAGE to the likely mechanism of sRAGE in the in RAGE signaling and of RAGE by sRAGE. may of sRAGE in humans. The receptor for advanced glycation end products (RAGE) 1The abbreviations used are: RAGE, receptor for advanced glycation end products; sRAGE, soluble RAGE; RT-PCR, reverse transcription PCR; LC-MS/MS, liquid chromatography tandem mass spectrometry; MALDI-MS, matrix-assisted laser desorption-ionization mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; PNGase F, N-glycosidase F; ECM, extracellular matrix.1The abbreviations used are: RAGE, receptor for advanced glycation end products; sRAGE, soluble RAGE; RT-PCR, reverse transcription PCR; LC-MS/MS, liquid chromatography tandem mass spectrometry; MALDI-MS, matrix-assisted laser desorption-ionization mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; PNGase F, N-glycosidase F; ECM, extracellular matrix. is a member of the immunoglobulin superfamily of cell surface receptors (1Neeper M. Schmidt A.M. Brett J. Yan J. is of extracellular a and a that is for RAGE-mediated signaling J. of of which advanced glycation end and in A.M. Yan Yan in humans, a cell signaling is in in the of the transcription factor J. J. M. J. Schmidt A.M. M. M. J. M. M. M. M. M. Schmidt A.M. Schmidt A.M. J. the RAGE J. Schmidt A.M. J. that to a such that RAGE is its In RAGE and its This is to the that RAGE is in is of chronic Alzheimer's and J. M. J. Schmidt A.M. Schmidt A.M. M. Schmidt A.M. J. M. M. J. Schmidt A.M. J. RAGE has a secreted soluble RAGE or sRAGE. a is secreted and acts as a sRAGE has been in a number of cell and animal models of RAGE-mediated successfully or RAGE signaling such as and Schmidt A.M. J. Yan Schmidt A.M. J. J. Schmidt A.M. J. M. J. Schmidt A.M. of the Yan J. J. Schmidt A.M. M. and cell and M. Schmidt A.M. sRAGE has been used in has been to biochemically characterize mouse sRAGE and the mechanism of its and M. J. found that in humans, sRAGE is produced by alternative splicing of RAGE mRNA. The of the and RAGE and sRAGE to carboxyl-terminal data that that mouse sRAGE is produced by carboxyl-terminal alternative the glycosylation and disulfide of sRAGE and a for sRAGE from mouse that a of sRAGE that the extracellular of sRAGE. from mouse from used as in by and was to the to J. in to of a and the was as was to the and was a and a The was was a of from the was and to The was to a and proteins a of a of sRAGE by mouse as J. and in from chromatography to as and to a The was the for the for the and, a of and as and of the chromatography was a liquid chromatography by was as J. of of sRAGE was as J. by and to mouse RAGE that sRAGE, or was mouse as J. or a mouse the the mouse RAGE RAGE found in the number to which was as for and for of for for and for and a for was and conditions as for of for for and for and a for of sRAGE for of sRAGE was and in the and and in the was in to the and of or of or of for a mass to The was used to the and a The reverse a high The was a of and from in to in data the for of the a and the data and as a The used to the data the from sRAGE and was a mass in in or of mouse RAGE was by for of sRAGE was of The was by to of sRAGE, the by in from was by reverse-phase chromatography a from to of and used to the the from a was as of by a disulfide of was to to that the mass to the mass of the Finally, from was and by to whether that the by a disulfide of of sRAGE was for in and was by the of of PNGase and the was for of sRAGE in was of for of was and the was for sRAGE PNGase by and as by the of of sRAGE from was to and as J. of of the to the and was to and by PNGase from sRAGE from mouse from used as in by and was to the to J. in to of a and the was as was to the and was a and a The was was a of from the was and to The was to a and proteins a of a of sRAGE by mouse as J. and in from chromatography to as and to a The was the for the for the and, a of and as and of the chromatography was a liquid chromatography by was as J. of of sRAGE was as J. by and to mouse RAGE that sRAGE, or was mouse as J. or a mouse the the mouse RAGE RAGE found in the number to which was as for and for of for for and for and a for was and conditions as for of for for and for and a for of sRAGE for of sRAGE was and in the and and in the was in to the and of or of or of for a mass to The was used to the and a The reverse a high The was a of and from in to in data the for of the a and the data and as a The used to the data the from sRAGE and was a mass in in or of mouse RAGE was by for of sRAGE was of The was by to of sRAGE, the by in from was by reverse-phase chromatography a from to of and used to the the from a was as of by a disulfide of was to to that the mass to the mass of the Finally, from was and by to whether that the by a disulfide of of sRAGE was for in and was by the of of PNGase and the was for of sRAGE in was of for of was and the was for sRAGE PNGase by and as by the of of sRAGE from was to and as J. of of the to the and was to and by PNGase from of sRAGE from used as for sRAGE sRAGE is found in mouse J. to was to by chromatography and the of mouse of to determine the of the mouse sRAGE that the of sRAGE was of the the of the mouse RAGE of mouse RAGE found in the number the is the may and to the which is in determine whether is the the of mouse sRAGE, the was and to The was to This to by the the and was as the was of the sRAGE the is to in for mouse sRAGE. sRAGE was or and to for is the of to the the of the The of the a the The the which our data of of mouse a is mouse sRAGE was and the by was used to determine which the indicated of and was used to that the of the in of is by a disulfide The disulfide sRAGE is in of by a disulfide the sRAGE from M. J. that sRAGE is a of alternative splicing of RAGE mRNA. The alternative is of the and results in a that a is for sRAGE. RAGE and sRAGE carboxyl-terminal whether mouse sRAGE is produced by alternative to alternative RAGE used mouse or a mouse as a in or to end of the RAGE as as the to the in and in to a that alternative splicing to that in humans. found of alternative RAGE to mouse RAGE and indicated by to and sRAGE from a that found by that our to alternative mouse RAGE is to of our to RAGE used in to mouse RAGE and indicated in mouse RAGE found in the data number mouse RAGE found in the data number used to successfully mouse mouse RAGE found in the data number used to successfully mouse mouse RAGE found in the data number and indicated in mouse RAGE found in the data number used to successfully mouse in a the mouse sRAGE used mouse sRAGE was or and by to that and the data from the data from the from our of the and was by the the carboxyl-terminal end of mouse sRAGE from our data is to the which the sRAGE from that of RAGE as by M. J. determine whether the mass of sRAGE sRAGE was and to mouse sRAGE the in our its the of the found that mass of mouse sRAGE a the that mouse sRAGE is and a from mouse sRAGE by indicated of of of of sRAGE. in a of characterize mouse sRAGE, its disulfide was used for the that in from mouse sRAGE to from to to from to by a disulfide the of and by the to that the by a disulfide a of the of was to to that the mass to of of was and by to determine whether in that the of the sRAGE has and found that in disulfide a disulfide as and of of RAGE glycosylation was by J. found a in RAGE the same is true for sRAGE has the same as determine whether mouse sRAGE has and mouse sRAGE was PNGase F, or a of the of sRAGE by that mouse sRAGE is sRAGE was to was which the that mouse sRAGE is sRAGE and that of used sRAGE to and In addition, mass of mouse sRAGE and PNGase and a in of to and the of of the sRAGE that to of the by was by in and PNGase data used the to the of that has a glycosylation and has a or is sRAGE was PNGase and to to mouse J. mouse sRAGE that a to for and from used in the to determine the of the data for reported in reported in reported in of mass and by PNGase to for the of to from PNGase reported in mass and by PNGase to for the of to from PNGase in a of sRAGE from used as for sRAGE sRAGE is found in mouse J. to was to by chromatography and the of mouse sRAGE. of to determine the of the mouse sRAGE that the of sRAGE was of the the of the mouse RAGE of mouse RAGE found in the number the is the may and to the which is in determine whether is the the of mouse sRAGE, the was and to The was to This to by the the and was as the was of the sRAGE the is to in sRAGE from M. J. that sRAGE is a of alternative splicing of RAGE mRNA. The alternative is of the and results in a that a is for sRAGE. RAGE and sRAGE carboxyl-terminal whether mouse sRAGE is produced by alternative to alternative RAGE used mouse or a mouse as a in or to end of the RAGE as as the to the in and in to a that alternative splicing to that in humans. found of alternative RAGE to mouse RAGE and indicated by to and sRAGE from a that found by that our to alternative mouse RAGE is to of our to RAGE the mouse sRAGE used mouse sRAGE was or and by to that and the data from the data from the from our of the and was by the the carboxyl-terminal end of mouse sRAGE from our data is to the which the sRAGE from that of RAGE as by M. J. determine whether the mass of sRAGE sRAGE was and to mouse sRAGE the in our its the of the found that mass of mouse sRAGE a the that mouse sRAGE is and a from mouse of characterize mouse sRAGE, its disulfide was used for the that in from mouse sRAGE to from to to from to by a disulfide the of and by the to that the by a disulfide a of the of was to to that the mass to of of was and by to determine whether in that the of the sRAGE has and found that in disulfide a disulfide as and of of RAGE glycosylation was by J. found a in RAGE the same is true for sRAGE has the same as determine whether mouse sRAGE has and mouse sRAGE was PNGase F, or a of the of sRAGE by that mouse sRAGE is sRAGE was to was which the that mouse sRAGE is sRAGE and that of used sRAGE to and In addition, mass of mouse sRAGE and PNGase and a in of to and the of of the sRAGE that to of the by was by in and PNGase data used the to the of that has a glycosylation and has a or a for sRAGE from mouse of which is in of sRAGE is in or J. This that sRAGE is to the extracellular matrix or cell and our that sRAGE has that sRAGE may to in the or cell in the or cell sRAGE may as a receptor by and from and cell surface RAGE and RAGE is to the that a in sRAGE that its to from the and the to has been that from the the M. J. Schmidt A.M. to determine whether the of is a of sRAGE of RAGE has been J. M. M. J. Schmidt A.M. The of in has to data that mouse sRAGE is produced by a mechanism from sRAGE, which is produced by alternative found for alternative RAGE in mouse or to a of RAGE, is likely that RAGE is to sRAGE. has been found to to sRAGE the J. in sRAGE from may that sRAGE of In to alternative sRAGE may by in a to that in mouse the the has been to in Yan M. Yan M. Schmidt A.M. J. sRAGE is in is likely that glycosylation is for the ability of sRAGE to bind its which in by J. a for sRAGE used a of glycosylation or by is to that is a the mass of mouse sRAGE as by and mass of sRAGE that is of sRAGE and sRAGE in is the of mouse sRAGE in and conditions, mouse sRAGE has been whether is in high mass or whether is sRAGE. our of sRAGE has been reported to Schmidt A.M. Yan Brett J. M. J. reported that RAGE to a results in a high mass by determine whether of in the mouse and is for the high mass in that sRAGE has which its in tissues. was found to that alternative splicing of RAGE to sRAGE in the mouse as was found for sRAGE. In the carboxyl-terminal of RAGE to the likely mechanism of sRAGE in the in RAGE signaling and of RAGE by sRAGE. may of sRAGE in humans. a for sRAGE from mouse of which is in of sRAGE is in or J. This that sRAGE is to the extracellular matrix or cell and our that sRAGE has that sRAGE may to in the or cell in the or cell sRAGE may as a receptor by and from and cell surface RAGE and RAGE is to the that a in sRAGE that its to from the and the to has been that from the the M. J. Schmidt A.M. to determine whether the of is a of sRAGE of RAGE has been J. M. M. J. Schmidt A.M. The of in has to The data that mouse sRAGE is produced by a mechanism from sRAGE, which is produced by alternative found for alternative RAGE in mouse or to a of RAGE, is likely that RAGE is to sRAGE. has been found to to sRAGE the J. in sRAGE from may that sRAGE of In to alternative sRAGE may by in a to that in mouse sRAGE. the the has been to in Yan M. Yan M. Schmidt A.M. J. sRAGE is in is likely that glycosylation is for the ability of sRAGE to bind its which in by J. a for sRAGE used a of glycosylation or by sRAGE. is to that is a the mass of mouse sRAGE as by and mass of sRAGE that is of sRAGE and sRAGE in is the of mouse sRAGE in and conditions, mouse sRAGE has been whether is in high mass or whether is sRAGE. our of sRAGE has been reported to Schmidt A.M. Yan Brett J. M. J. reported that RAGE to a results in a high mass by determine whether of in the mouse and is for the high mass in In that sRAGE has which its in tissues. was found to that alternative splicing of RAGE to sRAGE in the mouse as was found for sRAGE. In the carboxyl-terminal of RAGE to the likely mechanism of sRAGE in the in RAGE signaling and of RAGE by sRAGE. may of sRAGE in humans. and for