The transsulfuration pathway, through which homocysteine from the methionine cycle provides sulfur for cystathionine formation, which may subsequently be used for glutathione synthesis, has not heretofore been identified as active in mammary cells. Primary human mammary epithelial cells (HMEC’s) were labeled with 35S-methionine for 24 hours following pretreatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthionine sulfoximine. Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compound monochlorobimane to form a fluorescent glutathione-bimane conjugate. Comparison of fluorographic and autoradiographic images indicated that glutathione had incorporated 35S-methionine demonstrating that functional transsulfuration occurs in mammary cells. Pathway inhibitors reduced incorporation by roughly 80%. Measurement of glutathione production in HMEC’s treated with and without hydrogen peroxide and/or pathway inhibitors indicates that the transsulfuration pathway plays a significant role in providing cysteine for glutathione production both normally and under conditions of oxidant stress.

In this report, we describe the use of novel mass spectrometry instrumentation to identify a male-specific minor histocompatibility Ag restricted by HLA-A*0101 (A1-HY). This Ag has the sequence IVDC*LTEMY, where C* represents a cysteine disulfide bonded to a second cysteine residue. The core peptide sequence is found in the protein product of DFFRY, a Y chromosome gene not previously identified as the source of an HY Ag. The male-specific form of the peptide differs from its X chromosomal counterpart by the substitution of serine for the C* residue. Both peptides are expressed on the cell surface at 30 or fewer copies per cell. However, A1-HY-specific CTL recognize the DFFRY-derived peptide at a 1500-fold lower dose than the female homologue. Thus, these studies have identified a new source of HY epitopes and provide additional information about the influence of posttranslational modifications of class I-associated peptides on T cell recognition.

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C‐mannosylation is a type of protein glycosylation involving a covalently attached mannose sugar to the C2 carbon of an indole ring on tryptophan. Lipoprotein lipase (LPL), is an enzyme involved in lipid metabolism, hydrolysis of triglycerides in chylomicrons and very low‐density lipoproteins. LPL is mainly expressed in adipose tissue, muscle tissue, and macrophages, and it has been shown to contain a C‐mannosylated tryptophan in LPL‐overexpressing cell lines. Metabolic disorders, such as diabetes and obesity, leads to a deficiency of LPL function. LPL can be regulated at post‐transcriptional levels in a tissue specific manner that also depends on the nutritional state and hormonal level of the tissue. The goal of this project is to determine whether LPL is C‐mannosylated in adipose tissue from obese and lean Zucker rats. The Zucker fatty rat is a well‐established model of obesity and insulin resistance. Obese Zucker rats have high levels of lipids and cholesterol in their bloodstream, and their adipocytes are increased in size and number. LPL was isolated by immunoprecipitation (IP) using a monoclonal antibody specific for the LPL protein and by using heparin‐sepharose affinity chromatography. The isolated protein was analyzed by mass spectrometry utilizing the peptide mass fingerprint technique and low‐energy CID tandem MS/MS to identify C‐mannosylated tryptophan residues. The results of this experiment further the knowledge of the role that obesity plays in the post‐translational modification of LPL in adipose tissue.Support or Funding InformationNSF‐OIA‐1738707This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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