Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin

Fetuin, also known as alpha-2-Heremans Schmid glycoprotein (AHSG), belongs to some of the most abundant glycoproteins secreted into the bloodstream. In blood, fetuins exhibit functions as carriers of metals and small molecules. Bovine fetuin, which harbors 3 N-glycosylation sites and a suggested half dozen O-glycosylation sites, has been used often as a model glycoprotein to test novel analytical workflows in glycoproteomics. Here we characterize and compare fetuin in depth, using protein from three different biological sources: human serum, bovine serum, and recombinant human fetuin expressed in HEK-293 cells, with the aim to elucidate similarities and differences between these proteins and the post-translational modifications they harbor. Combining data from high-resolution native mass spectrometry and glycopeptide centric LC-MS analysis, we qualitatively and quantitatively gather information on fetuin protein maturation, N-glycosylation, O-glycosylation, and phosphorylation. We provide direct experimental evidence that both the human serum and part of the recombinant proteins are processed into two chains (A and B) connected by a single interchain disulfide bridge, whereas bovine fetuin remains a single-chain protein. Although two N-glycosylation sites, one O-glycosylation site, and a phosphorylation site are conserved from bovine to human, the stoichiometry of the modifications and the specific glycoforms they harbor are quite distinct. Comparing serum and recombinant human fetuin, we observe that the serum protein harbors a much simpler proteoform profile, indicating that the recombinant protein is not ideally engineered to mimic human serum fetuin. Comparing the proteoform profile and post-translational modifications of human and bovine serum fetuin, we observe that, although the gene structures of these two proteins are alike, they represent quite distinct proteins when their glycoproteoform profile is also taken into consideration

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin

Fetuin, also known as alpha-2-Heremans Schmid glycoprotein (AHSG), belongs to some of the most abundant glycoproteins secreted into the bloodstream. In blood, fetuins exhibit functions as carriers of metals and small molecules. Bovine fetuin, which harbors 3 N-glycosylation sites and a suggested half dozen O-glycosylation sites, has been used often as a model glycoprotein to test novel analytical workflows in glycoproteomics. Here we characterize and compare fetuin in depth, using protein from three different biological sources: human serum, bovine serum, and recombinant human fetuin expressed in HEK-293 cells, with the aim to elucidate similarities and differences between these proteins and the post-translational modifications they harbor. Combining data from high-resolution native mass spectrometry and glycopeptide centric LC-MS analysis, we qualitatively and quantitatively gather information on fetuin protein maturation, N-glycosylation, O-glycosylation, and phosphorylation. We provide direct experimental evidence that both the human serum and part of the recombinant proteins are processed into two chains (A and B) connected by a single interchain disulfide bridge, whereas bovine fetuin remains a single-chain protein. Although two N-glycosylation sites, one O-glycosylation site, and a phosphorylation site are conserved from bovine to human, the stoichiometry of the modifications and the specific glycoforms they harbor are quite distinct. Comparing serum and recombinant human fetuin, we observe that the serum protein harbors a much simpler proteoform profile, indicating that the recombinant protein is not ideally engineered to mimic human serum fetuin. Comparing the proteoform profile and post-translational modifications of human and bovine serum fetuin, we observe that, although the gene structures of these two proteins are alike, they represent quite distinct proteins when their glycoproteoform profile is also taken into consideration

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin

Fetuin, also known as alpha-2-Heremans Schmid glycoprotein (AHSG), belongs to some of the most abundant glycoproteins secreted into the bloodstream. In blood, fetuins exhibit functions as carriers of metals and small molecules. Bovine fetuin, which harbors 3 N-glycosylation sites and a suggested half dozen O-glycosylation sites, has been used often as a model glycoprotein to test novel analytical workflows in glycoproteomics. Here we characterize and compare fetuin in depth, using protein from three different biological sources: human serum, bovine serum, and recombinant human fetuin expressed in HEK-293 cells, with the aim to elucidate similarities and differences between these proteins and the post-translational modifications they harbor. Combining data from high-resolution native mass spectrometry and glycopeptide centric LC-MS analysis, we qualitatively and quantitatively gather information on fetuin protein maturation, N-glycosylation, O-glycosylation, and phosphorylation. We provide direct experimental evidence that both the human serum and part of the recombinant proteins are processed into two chains (A and B) connected by a single interchain disulfide bridge, whereas bovine fetuin remains a single-chain protein. Although two N-glycosylation sites, one O-glycosylation site, and a phosphorylation site are conserved from bovine to human, the stoichiometry of the modifications and the specific glycoforms they harbor are quite distinct. Comparing serum and recombinant human fetuin, we observe that the serum protein harbors a much simpler proteoform profile, indicating that the recombinant protein is not ideally engineered to mimic human serum fetuin. Comparing the proteoform profile and post-translational modifications of human and bovine serum fetuin, we observe that, although the gene structures of these two proteins are alike, they represent quite distinct proteins when their glycoproteoform profile is also taken into consideration

Reciprocal Regulation of C-Maf Tyrosine Phosphorylation by Tec and Ptpn22

<div><p>C-Maf plays an important role in regulating cytokine production in T_H cells. Its transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. In this study, we demonstrate that Tec kinase family member Tec, but not Rlk or Itk, is a tyrosine kinase of c-Maf and that Tec enhances c-Maf-dependent IL-4 promoter activity. This effect of Tec is counteracted by Ptpn22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by T_H17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on T_H cell-mediated immune responses.</p></div

Normal protein level and degree of tyrosine phosphorylation of c-Maf in Ptpn22-deficient T_H17 cells.

<p>(<b>A</b>) HEK 293T cells were transfected with pGL3-<i>Il21</i>-Luc, pRL-TK together with vectors expressing WT c-Maf, Tec, and/or Ptpn22. Luciferase activity was quantified 24 hours later and normalized according to Materials and Methods. The normalized luciferase activity obtained from cells transfected with empty expression vector was arbitrarily set as 1. The data shown are means ± SEM from three independent experiments. (<b>B & C</b>) Wild type (Ptpn22 WT) and Ptpn22 KO T_H cells were differentiated into T_H17 cells. The differentiated cells were re-stimulated with anti-CD3 antibody for 6 hr or 24 hr and the expression of IL-21 gene was measured by qPCR analysis (<b>B</b>). Mean and SEM values were obtained from three independent experiments. NS stands for not significant. Whole cell extract was harvested after re-stimulation with anti-CD3 for 24 hr, and subjected to immunoprecipitation with anti-c-Maf antibody or control antibody. The immunoprecipitant was then probed with 4G10 or anti-c-Maf antibody (<b>C</b>). Phosphorylation ratios of c-Maf are also shown.</p

Tyrosine phosphorylation at Tyr21/92/131 of c-Maf is critical for optimal IL-21 production in T_H17 cells.

<p>(<b>A</b>) Primary T_H cells were stimulated <i>in vitro</i> under T_H17 skewing conditions for 72 hours. Cells were lysed and then immunoprecipitated with anti-c-Maf or control IgG. The immunoprecipitant was probed with anti-p-Tyr and anti-c-Maf antibodies. (<b>B</b>) Primary T_H cells were stimulated <i>in vitro</i> under T_H17 skewing condition for 40 hours and transduced with retrovirus expressing c-Maf WT-FLAG or c-Maf Y3F-FLAG. Forty-eight hours later, transduced cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 24 hours. Cells were lysed and then immunoprecipitated with anti-c-Maf antibody. The immunoprecipitant was probed with 4G10 and anti-c-Maf antibodies. Phosphorylation ratios of c-Maf are also shown. (<b>C</b>) Primary T_H cells were stimulated <i>in vitro</i> under T_H2 and T_H17 skewing conditions for 40 hours and transduced with retrovirus expressing c-Maf WT-FLAG or Y3F-FLAG. Forty-eight hours later, transduced (GFP⁺) cells were sorted and re-stimulated with plate-bound anti-CD3 antibody for 24 hours. The levels of IL-4, IL-17 and IL-21 in supernatant were measured by ELISA in triplicate. The data shown are mean ± SEM from three independent experiments,* <i>P</i><0.05 and *** <i>P</i><0.001. (<b>D</b>) Retroviral transduced T_H17 cells described in (C) were sorted according to GFP expression, re-stimulated with PMA and ionomycin for 1 hour, and subjected to ChIP using primers derived from the <i>Il21</i> promoter.</p

Ptpn22 attenuates tyrosine phosphorylation of c-Maf and reduces c-Maf-dependent transactivation of the IL-4 reporter.

<p>(<b>A</b>) HEK 293T cells were transfected with plasmids expressing HA-c-Maf, Tec and/or Ptpn22. The transfected cells were lysed and then immunoprecipitated with anti-c-Maf antibody. The immunoprecipitant was then examined with Western blotting using 4G10 or c-Maf antibody. (<b>B</b>) HEK 293T cells were transfected with pGL3-<i>Il4</i>-Luc, pRL-TK, along with vectors expressing c-Maf and escalating levels Ptpn22. Luciferase activity was quantified 24 hours later and normalized according to Materials and Methods. The normalized luciferase activity obtained from cells transfected with empty expression vector was arbitrarily set as 1. (<b>C</b>) HEK 293T cells were transfected with pGL3-<i>Il4</i>-Luc, pRL-TK together with vectors expressing WT c-Maf, Tec, and/or Ptpn22. The normalized luciferase activity was calculated as in (B). (B) and (C) were performed in triplicate. The data shown are mean ± SEM from three independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001.</p

Tec enhances IL-4 promoter activity by facilitating the binding of c-Maf to the IL-4 promoter.

<p>(<b>A</b>) HEK 293T cells were transfected with pGL3-<i>Il4</i>-Luc and pRL-TK along with expression vectors for WT c-Maf, Y3F c-Maf, and/or Tec. The transfected cells were lysed after 24 hours and the luciferase activity was first normalized according to Materials and Methods and then against the value obtained with empty expression vector, which was arbitrarily set as 1. Each experiment was done in triplicate. The data shown are mean ± SEM from three independent experiments. ** <i>P</i><0.01. NS stands for not significant. (<b>B</b>) Nuclear extracts were prepared from HEK 293T cells expressing c-Maf and Tec, and subjected to EMSA using a 33 bp biotinylated probe derived from the MARE element (-31 to -64) within the IL-4 promoter (biotin-IL-4 probe). Unlabeled IL-4 probe (cold-IL-4 probe) and anti-c-Maf were added to the indicated lanes. A fraction of the nuclear extract used in lanes 2 and 3 was analyzed with Western blotting using anti-c-Maf (the lower panel).</p

Tec induces tyrosine phosphorylation of c-Maf.

<p>(<b>A</b>) HA-c-Maf was co-expressed with various C-terminal 3XFLAG-tagged Tec kinase members (Tec, Rlk or Itk) or a kinase dead Tec (Tec KD) in HEK 293T cells. Whole cell extract was harvested after 24 hours and immuno-precipitated (IP) with anti-c-Maf antibody. The immunoprecipitant was then immuno-blotted (IB) with anti-p-Tyr, anti-c-Maf and anti-FLAG antibodies. (<b>B</b>) Tec was co-expressed with EGFP-fused full-length, activation domain (AD), HINGE domain (HINGE), or DNA binding domain (DBD) of c-Maf in EL4 cells. The cells were lysed 24 hours after transfection and cell extract was immunoprecipitated with anti-EGFP antibody. The immunoprecipitant was then probed with 4G10 or anti-EGFP antibody. The various forms of c-Maf were marked with arrowheads. (<b>C</b>) Wild type c-Maf or various c-Maf mutants, in which tyrosine residues were converted to phenylalanine, were co-expressed with Tec in EL4 cells. The transfected cells were lysed 24 hours later and then immunoprecipitated with anti-c-Maf antibody. The immunoprecipitant was then probed with 4G10 or anti-c-Maf antibody. Y8F carries Y-to-F mutation at Tyr21/91/92/97/131/181/341/345. Phosphorylation ratios of c-Maf are also shown. (<b>D</b>) Naïve T_H cells were stimulated under T_H2 skewing conditions for 48 hours and then transduced with retrovirus expressing GFP alone (GFPRV mock) or along with murine Tec (GFPRV Tec) or lentivirus expressing shRNA specific for Tec (LV shTec). Forty-eight hours later, the transduced cells were re-stimulated with PMA/ionomycin (P+I) for 5 hr, lysed and immunoprecipitated with anti-c-Maf antibody. The immunoprecipitant was then probed with 4G10 or anti-c-Maf antibody. A fraction of un-precipitated extract was probed with anti-Tec and anti-Tubulin (the bottom two panels). Phosphorylation ratios of c-Maf are also shown.</p

Deficiency of Ptpn22 has little impact on the level and tyrosine phosphorylation of c-Maf in T_H2 cells.

<p>(<b>A</b>) Wild type (Ptpn22 WT) and Ptpn22 KO T_H cells were differentiated into T_H2 cells. The differentiated cells were re-stimulated with anti-CD3 antibody and the production of IL-4 was measured with ELISA. Mean and SEM values were obtained from twelve independent samples (n = 12). NS stands for not significant. (<b>B</b>) Primary T_H cells were isolated from WT or Ptpn22 KO mice and skewed under T_H1 or T_H2 conditions. Cells were rested (-) or re-stimulated with PMA/ionomycin (P+I) for 4 hr. Cell extract was harvested and probed with anti-c-Maf Ab or anti-α–Tubulin antibody. The density of each band shown was quantified with ImageJ. The band with the lowest density on each panel was arbitrarily set as 1. The c-Maf/α–Tubulin ratio was calculated by dividing the relative density of c-Maf by that of α–Tubulin. (<b>C</b>) Cell extract from stimulated WT and Ptpn22KO T_H2 cells described in (B) was also subjected to immunoprecipitation with anti-c-Maf antibody or control antibody. The immunoprecipitant was then probed with 4G10 or anti-c-Maf antibody. Phosphorylation ratios of c-Maf are also shown.</p