Specific Glutamine and Asparagine Residues of γ-S Crystallin Are Resistant to in Vivo Deamidation

Abstract

It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as γ-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from γ-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation. It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as γ-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from γ-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation. Probably the most common post-translational modification occurring in living systems involves nonenzymatic deamidation of glutamine and asparagine residues. This process occurs during the differentiation and/or aging of cells, resulting in the accumulation of glutamate and aspartate, respectively. Because deamidation results in the introduction of negative charges, it is possible that deamidation may play a major role in protein structural changes that are known to occur in development and aging. Furthermore, it has been hypothesized that deamidation may have a major effect upon the turnover rate of proteins (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar). Consistent with this hypothesis has been the observation that proteins that turn over rapidly generally have greater rates of deamidation, whereas proteins that are more stable have glutamine and/or asparagine residues that are more resistant to this process (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar).Perhaps the most stable proteins in the body are α, β, and γ crystallins found in the lens. Because of the inability of differentiated fiber cells to synthesize appreciable amounts of new protein, lens crystallins must remain relatively intact during the entire lifetime of the individual. The central, fetal-embryonic region of an aged human lens contains crystallins that were synthesized during the prenatal period of lens growth. Characterization of these crystallins should be a good test of the hypothesis that stable proteins, which contain glutamine and asparagine residues, have very low rates of deamidation. Previous studies have shown that from proteins of the human contains glutamine residues that are resistant to in Scholar). Because the and γ crystallins to a of lens proteins, and the of proteins in the of the it is important to asparagine and glutamine residues in this of lens proteins are relatively resistant vivo it is to of the and γ crystallins from the aged human lens a of post-translational that results in in and in the aged human a of and γ crystallins are it more to the of deamidation of asparagine and glutamine residues of these we to lens proteins from the fetal-embryonic region of aged human lenses and followed this by using synthetic to amidated and deamidated tryptic of γ-S synthetic have been as to the of amidated and deamidated Because of the of tryptic from of lens proteins, we have containing to the amidated and deamidated As an of possible deamidation occurring in the and γ crystallins during we have to possible amidated and deamidated of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from γ-S The results of demonstrate no detectable deamidation of these residues during the entire lifetime of lenses from aged human consistent with the hypothesis that very long-lived proteins contain glutamine and asparagine residues that are resistant to the deamidation human lenses and were from the The lenses were the lens the fetal-embryonic region of lens was by Scholar). This region of the lens was in and and was by and as Scholar). was as using as The protein was digested with of in of containing and of the resulting was a using of in peptide standards were to the resolution of of the to amidated deamidated tryptic of γ-S crystallin were followed by and resolution using the with of in to amidated and deamidated tryptic were synthesized by using peptide were using phase Characterization of by mass and was by the of synthetic peptide standards to tryptic fragments containing amidated deamidated forms of glutamine 92, glutamine 96, asparagine 143, and glutamine synthetic were as to the the phase resolution of these from the tryptic of proteins from the fetal-embryonic region of the aged human peptide in a new the of tryptic from the fetal-embryonic region of a human lens. of in was over a period of it was found that these the amidated and deamidated forms of the γ-S in with the of the amidated no was found with the of the deamidated of this spectral of the that it a of which was the as the of the amidated of The results of demonstrate that no detectable deamidation of asparagine in has during the the protein was a synthetic of γ-S were to the the of the amidated and deamidated forms of this a major that with the of the amidated of the were that the of the deamidated of these to the deamidated the by the was followed by resolution using a containing of as Previous results have shown these can with it possible to a of a phase by using containing that the amidated of was as a with the of the of the deamidated of this spectral of the that it a which is the as the of the amidated possible deamidation of glutamine The tryptic from the fetal-embryonic region of a lens was a phase using a of in over a period of the of is The the of the amidated of the the of the deamidated of and the the using a of from using a phase and of in over a period of the of is The the of the amidated of the of the deamidated of the resolution of tryptic from the lens region using an of in The be to the of the amidated and deamidated forms of γ-S and were containing as the that the to in a the of the amidated of spectral of this the of with and The to amidated γ-S with whereas the of is no to the of the deamidated of were no deamidation of the glutamine from the fetal-embryonic region of the possible deamidation of glutamine and glutamine using phase chromatography of a tryptic of proteins from the fetal-embryonic region of a lens. the of is The was with a phase using a of in over a period of The the of the amidated of the the of the deamidated of the the of the amidated of the the of the deamidated of the the phase chromatography of the in of in was over an of the of is The the of the amidated of the the of the deamidated of shown in the of to the of the amidated and deamidated forms of of γ-S crystallin was and using as an that a the of the amidated of this peptide with no the of the deamidated of the γ-S crystallin spectral of the in that it of which was the as the of the amidated of from γ-S phase chromatography of the in of in was over an of the of is The the of the amidated of the the of the deamidated of results of demonstrate that the fetal-embryonic region of a is no detectable deamidation of asparagine 143, glutamine glutamine 92, and glutamine from γ-S an the fetal-embryonic of lenses, and were shown to contain the γ-S in the amidated these results demonstrate the stability of of the asparagine and glutamine residues of γ-S crystallin in vivo deamidation during aging of the human nonenzymatic deamidation of asparagine and glutamine residues is to be an important in the turnover of proteins, it is possible that resistance to deamidation, in is a of long-lived Previous have the deamidation rate of proteins with as as and as as (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar). cells contain and structural proteins that rapidly (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google an observation consistent with the of deamidation in protein very is proteins that are extremely stable in vivo. to deamidation of such proteins the hypothesis that deamidation rates may play a role in rates of central, fetal-embryonic region of the aged human lens is the to in the nonenzymatic deamidation and protein stability of long-lived This region fiber cells that are studies a possible of proteins the central region of the lens it is generally that proteins this region were synthesized in the lifetime of the during the fetal-embryonic period of lens development. The has of the of old human lenses, in which the central fetal-embryonic region contains a relatively of proteins which have been It is hypothesized that the crystallins stability over the lifetime of the should contain asparagine and glutamine residues that are relatively resistant vivo deamidation. using synthetic to amidated and deamidated tryptic it is possible to the high pressure liquid chromatography to the forms from and from of this involves the of and in the Previous studies have that these with it possible to and the of the of in a tryptic of lens. it is that this can be to deamidation of asparagine and glutamine residues in aged it is that peptide fragments be tryptic studies using crystallin from proteins from the region of the aged human lens have that glutamine residues were resistant to deamidation Scholar). Because crystallin has a and from the the has the that a of the of crystallins contain glutamine and/or asparagine residues resistant to deamidation. The and γ crystallins of the human lens have and together the of proteins from the results of the demonstrate that of the major of the crystallin γ-S contains glutamine and asparagine residues that are extremely resistant vivo deamidation, consistent with the hypothesis that resistance to deamidation is a common to long-lived tryptic were that a glutamine asparagine This the of synthetic peptide standards development of the the synthetic this can be to and deamidation in tryptic fragments containing glutamine and/or asparagine mass spectral has been to the deamidation of glutamine and asparagine residues of γ-S crystallin from human lens Scholar). The results that glutamine and glutamine 170 deamidation during the aging The the with the results are it may be to in the of the the of lenses, whereas the possible deamidation of proteins from the fetal-embryonic region of old deamidation in proteins from the of the it is possible that deamidation may a greater of γ-S crystallin to be found in the of the protein by the are to a more of the of deamidation of these the the stability of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 of γ-S crystallin deamidation are such be important in the of deamidation during the aging of the of deamidation of and proteins has that protein as as the the asparagine may the deamidation. γ-S protein from aged human lens should an to the of protein upon deamidation, is known the of this Scholar). of this protein has that glutamine 92, glutamine 96, asparagine 143, and glutamine 170 are to deamidation such residues may be important may be in the of crystallins that has been hypothesized to be lens to the it is possible that of cells containing long-lived proteins may play an important role in the stability of proteins in vivo. to this the have upon that may the rates of test of this hypothesis be to the and rates of deamidation of crystallins in lens the in this it should be possible to the deamidation rates of major crystallin in to protein structural and/or that may in the resistance of glutamine and asparagine residues in vivo deamidation. Probably the most common post-translational modification occurring in living systems involves nonenzymatic deamidation of glutamine and asparagine residues. This process occurs during the differentiation and/or aging of cells, resulting in the accumulation of glutamate and aspartate, respectively. Because deamidation results in the introduction of negative charges, it is possible that deamidation may play a major role in protein structural changes that are known to occur in development and aging. Furthermore, it has been hypothesized that deamidation may have a major effect upon the turnover rate of proteins (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar). Consistent with this hypothesis has been the observation that proteins that turn over rapidly generally have greater rates of deamidation, whereas proteins that are more stable have glutamine and/or asparagine residues that are more resistant to this process (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar). the most stable proteins in the body are α, β, and γ crystallins found in the lens. Because of the inability of differentiated fiber cells to synthesize appreciable amounts of new protein, lens crystallins must remain relatively intact during the entire lifetime of the individual. The central, fetal-embryonic region of an aged human lens contains crystallins that were synthesized during the prenatal period of lens growth. Characterization of these crystallins should be a good test of the hypothesis that stable proteins, which contain glutamine and asparagine residues, have very low rates of deamidation. Previous studies have shown that from proteins of the human contains glutamine residues that are resistant to in Scholar). Because the and γ crystallins to a of lens proteins, and the of proteins in the of the it is important to asparagine and glutamine residues in this of lens proteins are relatively resistant vivo deamidation. it is to of the and γ crystallins from the aged human lens a of post-translational that results in in and in the aged human a of and γ crystallins are it more to the of deamidation of asparagine and glutamine residues of these we to lens proteins from the fetal-embryonic region of aged human lenses and followed this by using synthetic to amidated and deamidated tryptic of γ-S synthetic have been as to the of amidated and deamidated Because of the of tryptic from of lens proteins, we have containing to the amidated and deamidated As an of possible deamidation occurring in the and γ crystallins during we have to possible amidated and deamidated of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from γ-S The results of demonstrate no detectable deamidation of these residues during the entire lifetime of lenses from aged human consistent with the hypothesis that very long-lived proteins contain glutamine and asparagine residues that are resistant to the deamidation human lenses and were from the The lenses were the lens the fetal-embryonic region of lens was by Scholar). This region of the lens was in and and was by and as Scholar). was as using as The protein was digested with of in of containing and of the resulting was a using of in peptide standards were to the resolution of of the to amidated deamidated tryptic of γ-S crystallin were followed by and resolution using the with of in to amidated and deamidated tryptic were synthesized by using peptide were using phase Characterization of by mass and was by the of human lenses and were from the The lenses were the lens the fetal-embryonic region of lens was by Scholar). This region of the lens was in and and was by and as Scholar). was as using as The protein was digested with of in of containing and of the resulting was a using of in peptide standards were to the resolution of of the to amidated deamidated tryptic of γ-S crystallin were followed by and resolution using the with of in to amidated and deamidated tryptic were synthesized by using peptide were using phase Characterization of by mass and was by the of synthetic peptide standards to tryptic fragments containing amidated deamidated forms of glutamine 92, glutamine 96, asparagine 143, and glutamine synthetic were as to the the phase resolution of these from the tryptic of proteins from the fetal-embryonic region of the aged human peptide in a new the of tryptic from the fetal-embryonic region of a human lens. of in was over a period of it was found that these the amidated and deamidated forms of the γ-S in with the of the amidated no was found with the of the deamidated of this spectral of the that it a of which was the as the of the amidated of The results of demonstrate that no detectable deamidation of asparagine in has during the the protein was a synthetic of γ-S were to the the of the amidated and deamidated forms of this a major that with the of the amidated of the were that the of the deamidated of these to the deamidated the by the was followed by resolution using a containing of as Previous results have shown these can with it possible to a of a phase by using containing that the amidated of was as a with the of the of the deamidated of this spectral of the that it a which is the as the of the amidated of from using a phase and of in over a period of the of is The the of the amidated of the of the deamidated of the resolution of tryptic from the lens region using an of in The be to the of the amidated and deamidated forms of γ-S and were containing as the that the to in a the of the amidated of spectral of this the of with and The to amidated γ-S with whereas the of is no to the of the deamidated of were no deamidation of the glutamine from the fetal-embryonic region of the possible deamidation of glutamine and glutamine using phase chromatography of a tryptic of proteins from the fetal-embryonic region of a lens. the of is The was with a phase using a of in over a period of The the of the amidated of the the of the deamidated of the the of the amidated of the the of the deamidated of the the phase chromatography of the in of in was over an of the of is The the of the amidated of the the of the deamidated of shown in the of to the of the amidated and deamidated forms of of γ-S crystallin was and using as an that a the of the amidated of this peptide with no the of the deamidated of the γ-S crystallin spectral of the in that it of which was the as the of the amidated of from γ-S phase chromatography of the in of in was over an of the of is The the of the amidated of the the of the deamidated of results of demonstrate that the fetal-embryonic region of a is no detectable deamidation of asparagine 143, glutamine glutamine 92, and glutamine from γ-S an the fetal-embryonic of lenses, and were shown to contain the γ-S in the amidated these results demonstrate the stability of of the asparagine and glutamine residues of γ-S crystallin in vivo deamidation during aging of the human lens. synthetic peptide standards to tryptic fragments containing amidated deamidated forms of glutamine 92, glutamine 96, asparagine 143, and glutamine synthetic were as to the the phase resolution of these from the tryptic of proteins from the fetal-embryonic region of the aged human lens. the of tryptic from the fetal-embryonic region of a human lens. of in was over a period of it was found that these the amidated and deamidated forms of the γ-S in with the of the amidated no was found with the of the deamidated of this spectral of the that it a of which was the as the of the amidated of The results of demonstrate that no detectable deamidation of asparagine in has during the the protein was a synthetic of γ-S were to the the of the amidated and deamidated forms of this a major that with the of the amidated of the were that the of the deamidated of these to the deamidated the by the was followed by resolution using a containing of as Previous results have shown these can with it possible to a of a phase by using containing that the amidated of was as a with the of the of the deamidated of this spectral of the that it a which is the as the of the amidated the resolution of tryptic from the lens region using an of in The be to the of the amidated and deamidated forms of γ-S and were containing as the that the to in a the of the amidated of spectral of this the of with and The to amidated γ-S with whereas the of is no to the of the deamidated of were no deamidation of the glutamine from the fetal-embryonic region of the lens. As shown in the of to the of the amidated and deamidated forms of of γ-S crystallin was and using as an that a the of the amidated of this peptide with no the of the deamidated of the γ-S crystallin spectral of the in that it of which was the as the of the amidated of from γ-S The results of demonstrate that the fetal-embryonic region of a is no detectable deamidation of asparagine 143, glutamine glutamine 92, and glutamine from γ-S an the fetal-embryonic of lenses, and were shown to contain the γ-S in the amidated these results demonstrate the stability of of the asparagine and glutamine residues of γ-S crystallin in vivo deamidation during aging of the human lens. nonenzymatic deamidation of asparagine and glutamine residues is to be an important in the turnover of proteins, it is possible that resistance to deamidation, in is a of long-lived Previous have the deamidation rate of proteins with as as and as as (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar). cells contain and structural proteins that rapidly (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google an observation consistent with the of deamidation in protein very is proteins that are extremely stable in vivo. to deamidation of such proteins the hypothesis that deamidation rates may play a role in rates of central, fetal-embryonic region of the aged human lens is the to in the nonenzymatic deamidation and protein stability of long-lived This region fiber cells that are studies a possible of proteins the central region of the lens it is generally that proteins this region were synthesized in the lifetime of the during the fetal-embryonic period of lens development. The has of the of old human lenses, in which the central fetal-embryonic region contains a relatively of proteins which have been It is hypothesized that the crystallins stability over the lifetime of the should contain asparagine and glutamine residues that are relatively resistant vivo deamidation. using synthetic to amidated and deamidated tryptic it is possible to the high pressure liquid chromatography to the forms from and from of this involves the of and in the Previous studies have that these with it possible to and the of the of in a tryptic of lens. it is that this can be to deamidation of asparagine and glutamine residues in aged it is that peptide fragments be tryptic studies using crystallin from proteins from the region of the aged human lens have that glutamine residues were resistant to deamidation Scholar). Because crystallin has a and from the the has the that a of the of crystallins contain glutamine and/or asparagine residues resistant to deamidation. The and γ crystallins of the human lens have and together the of proteins from the results of the demonstrate that of the major of the crystallin γ-S contains glutamine and asparagine residues that are extremely resistant vivo deamidation, consistent with the hypothesis that resistance to deamidation is a common to long-lived tryptic were that a glutamine asparagine This the of synthetic peptide standards development of the the synthetic this can be to and deamidation in tryptic fragments containing glutamine and/or asparagine mass spectral has been to the deamidation of glutamine and asparagine residues of γ-S crystallin from human lens Scholar). The results that glutamine and glutamine 170 deamidation during the aging The the with the results are it may be to in the of the the of lenses, whereas the possible deamidation of proteins from the fetal-embryonic region of old deamidation in proteins from the of the it is possible that deamidation may a greater of γ-S crystallin to be found in the of the protein by the are to a more of the of deamidation of these the the stability of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 of γ-S crystallin deamidation are such be important in the of deamidation during the aging of the of deamidation of and proteins has that protein as as the the asparagine may the deamidation. γ-S protein from aged human lens should an to the of protein upon deamidation, is known the of this Scholar). of this protein has that glutamine 92, glutamine 96, asparagine 143, and glutamine 170 are to deamidation such residues may be important may be in the of crystallins that has been hypothesized to be lens to the it is possible that of cells containing long-lived proteins may play an important role in the stability of proteins in vivo. to this the have upon that may the rates of test of this hypothesis be to the and rates of deamidation of crystallins in lens the in this it should be possible to the deamidation rates of major crystallin in to protein structural and/or that may in the resistance of glutamine and asparagine residues in vivo deamidation. Because nonenzymatic deamidation of asparagine and glutamine residues is to be an important in the turnover of proteins, it is possible that resistance to deamidation, in is a of long-lived Previous have the deamidation rate of proteins with as as and as as (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google Scholar). cells contain and structural proteins that rapidly (1Robinson A.B. Rudd C.J. Curr. Top. Cell. Regul. 1974; 8: 248-295Google an observation consistent with the of deamidation in protein very is proteins that are extremely stable in vivo. to deamidation of such proteins the hypothesis that deamidation rates may play a role in rates of vivo. The central, fetal-embryonic region of the aged human lens is the to in the nonenzymatic deamidation and protein stability of long-lived This region fiber cells that are studies a possible of proteins the central region of the lens it is generally that proteins this region were synthesized in the lifetime of the during the fetal-embryonic period of lens development. The has of the of old human lenses, in which the central fetal-embryonic region contains a relatively of proteins which have been It is hypothesized that the crystallins stability over the lifetime of the should contain asparagine and glutamine residues that are relatively resistant vivo deamidation. using synthetic to amidated and deamidated tryptic it is possible to the high pressure liquid chromatography to the forms from and from of this involves the of and in the Previous studies have that these with it possible to and the of the of in a tryptic of lens. it is that this can be to deamidation of asparagine and glutamine residues in aged it is that peptide fragments be tryptic Previous studies using crystallin from proteins from the region of the aged human lens have that glutamine residues were resistant to deamidation Scholar). Because crystallin has a and from the the has the that a of the of crystallins contain glutamine and/or asparagine residues resistant to deamidation. The and γ crystallins of the human lens have and together the of proteins from the lens. The results of the demonstrate that of the major of the crystallin γ-S contains glutamine and asparagine residues that are extremely resistant vivo deamidation, consistent with the hypothesis that resistance to deamidation is a common to long-lived tryptic were that a glutamine asparagine This the of synthetic peptide standards development of the the synthetic this can be to and deamidation in tryptic fragments containing glutamine and/or asparagine residues. mass spectral has been to the deamidation of glutamine and asparagine residues of γ-S crystallin from human lens Scholar). The results that glutamine and glutamine 170 deamidation during the aging The the with the results are it may be to in the of the the of lenses, whereas the possible deamidation of proteins from the fetal-embryonic region of old deamidation in proteins from the of the it is possible that deamidation may a greater of γ-S crystallin to be found in the of the protein by the are to a more of the of deamidation of these residues. the the stability of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 of γ-S crystallin deamidation are such be important in the of deamidation during the aging of the of deamidation of and proteins has that protein as as the the asparagine may the deamidation. γ-S protein from aged human lens should an to the of protein upon deamidation, is known the of this Scholar). of this protein has that glutamine 92, glutamine 96, asparagine 143, and glutamine 170 are to deamidation such residues may be important may be in the of crystallins that has been hypothesized to be lens Scholar). to the it is possible that of cells containing long-lived proteins may play an important role in the stability of proteins in vivo. to this the have upon that may the rates of test of this hypothesis be to the and rates of deamidation of crystallins in lens the in this it should be possible to the deamidation rates of major crystallin in to protein structural and/or that may in the resistance of glutamine and asparagine residues in vivo deamidation. The and the of