the cellular thiol - disulfide redox environment is defined by protein thiols ( psh ) and disulfides ( psox ) as well as low molecular weight thiols and disulfides . in mammalian cells ,
by far the most abundant low molecular weight sulfhydryl molecule is glutathione ( gsh ) . together with its disulfide ( gssg ) , this pair is often referred to as the cellular thiol - disulfide redox buffer .   in the cytosol of eukaryotic cells , glutathione is highly reducing with a ratio of gsh to gssg of at least 3,000 [ 1 , 2 ] , and consequently the majority of protein cysteines are found as psh .
the high concentrations of psh and gsh in this compartment are important in the cellular defense against thiol oxidants , during thiol - disulfide stress , formation of mixed disulfides between protein and glutathione ( pssg ) serves as a mechanism for protecting psh and gsh from irreversible oxidation .
in contrast to cytosolic proteins , secretory proteins often contain disulfide bonds , and the glutathione redox pool in the secretory compartments of the cell is found to be considerably more oxidizing than the cytosolic pool .
disulfide bond formation is an essential step for the correct folding of many secretory proteins , and in eukaryotic cells their folding and assembly takes place in the endoplasmic reticulum ( er ) . in this compartment , molecular chaperones and enzymes for disulfide bond formation and glycosylation support protein folding .
the maintenance of a proper er redox environment is crucial for the folding of secretory proteins . if the redox environment becomes too reducing , the formation of disulfide bonds is hampered .
if too oxidizing , folding intermediates with nonnative disulfide bonds can accumulate . a number of oxidoreductases , which may have different functions and/or substrate or tissue specificities in the assistance of folding secretory proteins ,
the best characterized oxidoreductase is protein disulfide isomerase ( pdi ) , which introduces , reduces , and reorganizes disulfide bonds in a broad variety of substrate proteins .
the oxidative pathway remains unresolved , but pdi may be reoxidized by a number of enzymes including pdi peroxidases , gpx7 and gpx8 , peroxiredoxin 4 , and the flavoprotein ero1 ( endoplasmic reticulum oxidoreductin 1 ) , for review see [ 10 , 11 ] .
professional secretory cells are specialized in producing secretory proteins and are characterized by their abundant er .
one example is the terminally differentiated b cell , also referred to as plasma cell , which secretes enormous amounts of antibodies , that is , immunoglobulins ( ig ) .
while resting b cells do not secrete antibody , they do express a membrane - bound ig on their cell surface as a subunit of the b cell receptor , which upon binding of antigen activates a signaling cascade that can lead to differentiation into antibody - secreting plasma cells .
the differentiation is accompanied by many morphological changes to accommodate production of large amounts of secreted antibody .
this includes a general increase in cell volume with a preferential expansion of the er . in addition , the differentiation is accompanied by dramatic changes in the proteome of the cell [ 13 , 14 ] ; as expected , the er proteins are significantly up - regulated .
igm is typically secreted as disulfide - linked pentamers or hexamers of a subassembly consisting of two identical heavy chains (  ) and two light chains (  ) .
the pentameric holoprotein in addition contains a j - chain , which the hexamer does not . as each subassembly contains 16 disulfide bonds and the j - chain contributes 4 disulfide bonds ,
ros production is increased during b cell differentiation and counterbalanced by a strong antioxidant response .
we set out to investigate how this enormous load on the secretory machinery affects the global thiol - disulfide environment of the b cell .
we have applied a previously developed method for quantitative determination of the absolute levels of psh , psox , and pssg on all cellular proteins ( including membrane proteins ) in cultured mammalian cells , and combined these data with quantifications of gsh and gssg in the same cells . in this way
, we have obtained a picture of the global changes in cellular thiol - disulfide redox status during differentiation of the resting b cell into an antibody - secreting plasma cell .
quantitative studies of the cellular redox status involve a variety of technical challenges due to the reactive nature of the sh group .
great care must be taken to avoid artificial air oxidation and to eliminate cross - reactivity between the thiol and disulfide specific reagents , which can otherwise lead to deceptive conclusions . by applying a previously developed technology that carefully considers these technical pitfalls
, we can quantitatively determine the cellular levels of total sulfhydryl equivalents in low molecular mass thiols and in protein .
the key features of the experimental approach are illustrated in figure 1 . to avoid perturbation of the cellular thiol - disulfide status during cell lysis and sample preparation ,
cells were acidified by the addition of tca to a final concentration of 10% , resulting in immediate protein denaturation and precipitation .
this combination of rapid trapping and deprotonation simultaneously unfolds redox enzymes , some of which have low thiol pka and are fairly acid - stable , and quenches generic thiols by protonation . to fully exploit the strength of our approach , we did not facs - sort cells before analysis , nor did we homogenize and fractionate cells .
the tca pellets were solubilized by sonicating in appropriate buffers with high concentrations of sds or urea to quantify the different sulfhydryl species in all cellular proteins including membrane proteins .
psh and psox levels were determined with a highly sensitive hplc assay based on the thiol quantification agent 4-dps .
the total value of protein cysteines ( total ps ) was calculated by the addition of psh and psox and to verify the method , this value was also determined experimentally . for experiments performed on resting b cells , there is an excellent agreement between the experimentally determined value and the calculated sum of the experimentally determined psh and psox ( data not shown )
. finally , pssg levels were selectively quantified by the use of the thiol derivatization agent sbd - f .
the sbd - gs derivative is highly fluorescent and can be quantified specifically due to its unique retention time in an hplc chromatogram .
in addition to protein sulfhydryls , the total protein content of each sample was determined and used as a common denominator to compare the individual samples .
this is a crucial step as it eliminates any bias from a possible uneven division of the tca pellet into fractions .
furthermore , the requirement for a common denominator in this study is particularly important , as it also eliminates any bias due to morphological differences between cell samples .
the total protein content was quantified using a method based on complete hydrolysis in hcl followed by quantification of released amino acids with ninhydrin .
this constitutes a highly reproducible and sensitive method and , with a proper standard , it yields numbers that can be calibrated to  amino acids in protein .  furthermore , the ninhydrin assay is independent of protein solubility and hence includes both soluble and membrane proteins .
thus , all the following data will be shown as sulfhydryl per amino acid ( sh / aa ) . as a model for b cell differentiation , we used a previously established system based on the murine b cell lymphoma 1.29 which can be induced by lipopolysaccharide ( lps ) to secrete igm [ 13 , 19 ] . to obtain a well - defined reference point for the differentiation of b cells into plasma cells
, we quantified the thiol - disulfide status in uninduced b cells to obtain a reference point for differentiation into plasma cells .
cells were seeded to a density of 0.2  10  cells / ml , and samples for redox quantification were taken each day during the next four days . although cell density increased considerably during this period , the protein redox state remained constant throughout the experiment ( figures 2(a ) and 2(b ) ) .
in addition , levels of pssg , gssg , and gsh remained constant ( data not shown ) , and we concluded that the global thiol - disulfide status is independent of cell density .
accordingly , the mean value of data obtained for each of the redox species during the four days was calculated , and the relative distributions of the different protein and glutathione sulfhydryl equivalents are given in table 1 . from these data , we concluded that the vast majority of cellular sulfhydryl equivalents exists in the reduced thiol form with only 5% and 9% of the ps and gs equivalents engaged in disulfide bond formation , respectively .
together , these data describe the total thiol - disulfide environment of resting b cells , and we used them as reference point for studying the differentiation into antibody - secreting plasma cells . in the remaining part of this study
interestingly , the distribution of thiol and disulfide equivalents in resting b cells is very similar to that of hek ( human embryonic kidney ) cells , where 6% of the ps equivalents were found as psox and 8.5% of the gs equivalents were found as gssg . in addition , our observation that cellular pssg levels are extremely low is supported by results in hek and hela cells .
it should be mentioned that although cells are grown in the presence of -mercaptoethanol , which was detected in the tca supernatant and also in very small amounts in the tca pellet , it did not interfere with the glutathione or pssg measurements because fluorescent -mercaptoethanol derivatives were separated efficiently from gs derivatives by hplc ( data not shown ) .
resting 1.29 cells were treated with lps to induce differentiation into antibody - secreting plasma cells .
samples were prepared for global thiol quantification after 1 , 2 , 3 , and 4 days of lps treatment . from the sh / aa of total ps equivalents ( figure 3(a ) ) , we find that the frequency of cysteine residues in proteins is fairly constant ( ~2% ) throughout the differentiation .
this is in excellent agreement with the experimentally determined values for other mammalian cell lines as well as calculated values for eukaryotes in general hansen [ 3 , 20 ] .
the sh / aa for total ps was expected to be largely unaffected during differentiation as the igm monomer has a cysteine frequency of 2.5% ( igm has 1,540 amino acids , of which 38 are cysteines ) .
although the absolute value of total ps equivalents remained unchanged throughout the experiment , the distribution of protein thiols and disulfides was influenced by the lps - induced cell differentiation .
the percentage of protein thiols engaged in disulfide bond formation remained largely unaffected for two days after lps induction , but at day three the psox values had doubled , and a total increase by a factor of 3.3 was found at day four ( figure 3(b ) ) .
likewise , after a lag time of two days , on day three pssg had increased by a factor of 2.2 , but in contrast to psox , the pssg value did not increase further on day four ( figure 3(c ) ) .
interestingly , the ratio of pssg to psox remained essentially unchanged throughout the experiment ( figure 3(d ) ) .
quantification of soluble glutathione equivalents revealed that the absolute concentrations of gssg remained largely unaffected ( figure 4(a ) ) , but the fraction of oxidized gs equivalents ( gs in gssg ) relative to total gs equivalents ( total gs ) increased from 8.8% to 14.5% ( figure 4(b ) ) .
this was caused by a gradual decrease in gsh ( figure 4(a ) ) resulting in an overall decrease of 56% in ( total gs ) at day 4 compared to day 0 .
the gs equivalents were not recovered as pssg , which throughout the study remained a minute fraction of total gs equivalents ( figure 4(c ) ) . to rule out that the decrease in intracellular gs was caused by an increase in dying cells , the level of apoptotic and necrotic cells was measured using flow cytometry and staining with the cell - impermeable dye propidium iodide ( pi ) .
although the fraction of viable ( pi - negative ) cells decreased during the differentiation by a factor of 1.5 ( supplementary material figure 1 available online at http://dx.doi.org/10.1155/2013/898563 ) , it could not account for our observed decrease in intracellular glutathione ( see supplementary material text ) . as the decrease in intracellular gs was not explained by an increase in cell death , we measured whether gs equivalents were secreted by the cells .
no glutathione was detectable in a blank medium sample ( data not shown ) ; media from uninduced cells , however , contained measurable amounts of glutathione , and the values increased significantly on day three and four after induction ( figure 4(d ) ) .
it should be noted that these numbers are only rough estimates and that the values are most likely underestimated due to extracellular gs degradation catalyzed by -glutamyl transferase . from the data shown in figures 4(a ) and 4(d ) , the increase in extracellular gs / aa equivalents at day 4 relative to day 0 was calculated to ( 0.23  0.1 )    10 while the decrease in intracellular gs / aa was calculated to ( 0.46  0.05 )    10 .
as the two numbers are in the same size range , it is possible that the differentiating cells secrete glutathione ( details are given in supplementary material text ) .
the transformation of resting b cells into antibody - secreting plasma cells involves an extensive expansion of the er   and both er resident proteins and proteins involved in redox balance are up - regulated linearly during differentiation . how cells cope with this sudden increase in secretory activity has been the subject of numerous studies [ 22 , 23 ] .
this study , for the first time , provides a quantitative overview of the cellular thiol - disulfide status during differentiation of resting b cells into antibody - secreting plasma cells .
we applied a previously developed method to quantify soluble gsh and gssg as well as protein thiols and disulfides in cells .
importantly , the method includes both soluble and membrane proteins , which precludes bias due to morphological changes during b cell differentiation .
we find that the differentiation process affects the global protein thiol - disulfide status with an increase factor of 3.3 in the fraction of oxidized protein thiols at day 4 of differentiation , compared to day 0 .
the effects on the glutathione redox status are less significant with an increase factor of 1.6 in the fraction of oxidized gs equivalents .
the changes in glutathione redox state were caused by a general depletion of gs equivalents from differentiating b cells .  
the differentiation of b cells into plasma cells has been studied in great detail at the proteomic level [ 13 , 14 ] .
, the cell prepares by ensuring that metabolic capacity and secretory machinery can cope with the mass production of antibody molecules . we did not find any change in protein thiol - disulfide status until the third day of activation , when the fraction of psox increased by a factor of 2.2 .
the kinetics for the change in protein redox state were identical to the kinetics for igm production .
our results suggest that a general expansion of the er does not affect protein redox status until an extensive production of cargo proteins is initiated .
the increase of igm production at day three is possibly preempted by a process known as  proactive  unfolded protein response ( proactive upr ) .
the upr is a stress signaling process that is initiated when unfolded polypeptides accumulate in the er . this process leads to an up - regulation of er chaperones and folding enzymes which prevents er stress . while unfolded protein stress ( or er stress ) is not involved in the initial expansion of the er in professional secretory cells [ 13 , 14 ] , it is essential for b cell differentiation [ 26 , 27 ] .
upr - induced oxidases such as ero1 may facilitate the change in protein redox state on the third day of differentiation to initiate disulfide - dependent igm polymerization and its subsequent secretion .
initially gssg was thought to provide oxidizing equivalents for disulfide bond formation , but after identification of the ero1 proteins this hypothesis was discarded . instead , gsh
now is considered to be involved in the isomerization of nonnative disulfide bonds [ 2830 ] to consume excess oxidizing equivalents produced by the ero1 proteins   and to activate ero1 by reducing its regulatory disulfides .
consequently , the abundance of gssg in the er is altogether assumed to be at least partially caused by ero1 activity . the mechanisms by which the er maintains its gsh / gssg redox balance are unknown .
excess gssg could be reduced by an er resident glutathione reductase , it could be transported to the cytosol for reduction or it could be secreted from the cells . during b cell differentiation ero1 is up - regulated by factors of 3.0 and 2.4 at day 3 and 4 , respectively , and consequently we expected gssg levels to increase .
surprisingly , gssg levels remained constant throughout differentiation , but the overall cellular glutathione redox status did become more oxidizing gradually ( figure 4(a ) ) .
this was the result of a depletion of total gs equivalents at the expense of gsh ( figure 4(b ) ) .
there are three possible explanations for the decrease in total intracellular gs , ( 1 ) differentiation of b cells leads to secretion of gs equivalents , ( 2 ) gsh is irreversibly oxidized to sulfinic or sulfonic acids , which is not detected by the quantification method , and ( 3 ) gs equivalents are released by the fraction of pi positive cells .
we found an increase in extracellular gs on days 3 and 4 of the differentiation that was of about the same magnitude as the intracellular decrease ( figure 4(d ) ) .
this result supports model ( 1 ) and suggests that gsh converted to gssg by the up - regulation of ero1 proteins and is exported to the media .
this export could serve as a mechanism for relieving cells from any oxidative load caused by up - regulation of ero1 proteins .
we can , however , not make any certain conclusions regarding the cause of intracellular gs depletion , as the levels of extracellular gs could in principle be explained by the fraction of pi - positive cells releasing their intracellular gs content to the media ( see supplementary material text ) . due to
the extremely reducing glutathione redox potential of the cytosol , the vast majority of pssg is expected to be found in the oxidizing compartments of the cell . in a study of liver microsomes ,
50% of the gs equivalents were found as pssg , suggesting that a major fraction of the glutathione in the er is associated with protein .
however , this fraction was subsequently estimated to be significantly lower ( i.e. , less than 2% pssg ) based on whole - cell quantification with the assumption that no pssg is found in the cytosol . under the same assumption ; that is , that all pssg equivalents are found in the er and that the concentrations of gs equivalents are the same in all cellular compartments , we can estimate that the maximal fraction of pssg is relative to total gs equivalents in the er , in fully differentiated b cells .
the er volume is reported to constitute at least 10% of total cell volume in antibody - secreting b cells   and , accordingly , maximally 7% ( 0.7%/10% ) of the gs equivalents in er are found as pssg .
thus , even in highly active secretory cells , pssg only constitutes a minor fraction of total gs equivalents of the er . these results support the notion that the er glutathione redox environment is more reducing than previously assumed .
it is generally assumed that the level of gssg is critical for the amounts of pssg formed .
interestingly , we found that the ratio of pssg to psox was independent of differentiation ( figure 3(d ) ) , suggesting that the oxidizing compartments of the cell maintain a constant level of pssg , and consequently , that the er glutathione redox status is tightly regulated throughout differentiation .
this may be explained by the activation of oxidative stress during the early stage of b cell differentiation and followed by a strong antioxidant response [ 16 , 36 ] .
maintenance of a proper er glutathione redox environment can be a crucial factor in securing the correct folding of igm . in this study
, we have for the first time characterized the changes in thiol - disulfide state during differentiation of b cells .
in general , the differentiation does not cause massive thiol - disulfide stress to the cells .
the steady state levels of pssg are maintained at very low levels , even in fully differentiated cells , and the overall protein redox state is not affected until late in differentiation , when large - scale igm production has started and the er stress response has been activated .
1.29 cells were maintained in suspension as described in   and after 2 days , cell culture medium was replaced by fresh medium . for differentiation
, cells were cultured in the presence of 20  g / ml lps ( sigma ) .
three independent cultures were induced with lps and samples were taken after 1 , 2 , 3 , or 4 days of differentiation . as a control ,
samples from 3 independent cultures grown without lps were collected as well . each day after lps activation , samples were collected for flow cytometric analysis .
cells were stained with pi ( sigma ) and flow cytometry data were obtained with facscalibur ( bd biosciences ) and analyzed using cellquest software ( bd biosciences ) .
cells were harvested by centrifugation followed by a wash step with 10  ml dulbecco 's 1x pbs ( paa laboratories ) to eliminate traces of protein from the media .
cells were resuspended in 1  ml ice - cold 10% ( w / v ) tca and incubated on ice for 30 minutes followed by centrifugation .
the supernatant , used for quantification of gsh and gssg , was immediately frozen in n2 and kept at 80c until later use .
the tca pellet , used for protein thiol quantification , was washed in 10% tca by four cycles of sonication and centrifugation . before the final centrifugation step , the suspension
was divided in four to quantify psh , psox , pssg , and total ps .
the psh , pssg , and total ps samples were immediately frozen in n2 and kept at 80c until later use .
the psox sample was directly solubilized and alkylated by sonicating the pellet in 500  l of 5% sds , 1  mm edta , 20  mm nem ( sigma ) in 0.5  m tris - cl ph 8.3 .
the alkylation was allowed to proceed for at least 20  min before the sample was transferred to 80c .
thiol and disulfide species in tca supernatant and tca pellet fractions were quantified as described in .
briefly , psh samples were solubilized in 5% sds , 1  mm edta in 0.4  m sodium citrate , ph 4.5 and protein thiols were quantified by the addition of 4-dps ( sigma ) to a final concentration of 0.5  mm followed by hplc analysis . for quantification of psox , samples alkylated with nem were reduced by the addition of bh ( sigma ) to a final concentration of 3.3% ( w / v ) .
prior to quantification with 4-dps , bh was destroyed by the addition of hcl , as described in .
total ps was quantified by solubilizing the pellet in 5% sds , 1  mm edta , and 0.5  m tris - cl ph 8.3 , reducing all thiols with bh , followed by thiol quantification with 4-dps .
the pssg sample was solubilized in 6  m urea , 8  mm edta , and 200  mm bicine ph 9.2 .
disulfides were reduced with 2.1  mm thp ( calbiochem ) and thiols were derivatized by the addition of 6.4  mm sbd - f ( fluka ) for 1 hour at 60c . to compare samples , total protein content was determined by using a ninhydrin based assay . an hplc assay based on thiol derivatization with n-(1-pyrenyl)maleimides ( fluka ) was used to quantify gssg and total gs ( gsh + gssg ) from the supernatant as described in .
for quantification of secreted glutathione , proteins in media from harvested cells were precipitated by the addition of tca to 10% ( v / w ) and incubated on ice for 30 minutes followed by centrifugation .
 plasma cells produce and secrete massive amounts of disulfide - containing antibodies . to accommodate this load on the secretory machinery , the differentiation of resting b cells into antibody - secreting plasma cells 
 is accompanied by a preferential expansion of the secretory compartments of the cells and by an up - regulation of enzymes involved in redox regulation and protein folding . 
 we have quantified the absolute levels of protein thiols , protein disulfides , and glutathionylated proteins in whole cells . 
 the results show that while the global thiol - disulfide state is affected to some extent by the differentiation , steady - state levels of glutathionylated protein thiols are less than 0.3% of the total protein cysteines , even in fully differentiated cells , and the overall protein redox state is not affected until late in differentiation , when large - scale igm production is ongoing . 
 a general expansion of the er does not affect global protein redox status until an extensive production of cargo proteins has started . 