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    Researchers identify new protein with key role in antibody assembly

    Linda Hendershot and Yuichiro Shimizu

    St. Jude investigators identified a new protein at work inside infection-fighting plasma cells that plays a critical role in quality control during antibody production.

    The protein, named pERp1, functions only in B lymphocytes and plasma cells, the immune cells where antibodies that fight infection and other threats are made. Without pERp1, plasma cells struggle to make the chemical bonds essential for proper antibody assembly and functioning, researchers reported.

    But investigators said the predicted structure of pERp1 does not resemble any of the approximately 20 other enzymes at work in the protein-folding machinery that helps mammalian cells make the same chemical bonds, known as disulfide bonds. Researchers have also not completely ruled out the possibility that pERp1 is not the actual enzyme that catalyzes the formation of these bonds but that it works indirectly with one of the known enzymes. Writing in a recent edition of Proceedings of the National Academy of Sciences, the scientists noted that pERp1 might be a previously unidentified member of a completely different family of proteins, known as chaperone proteins. Chaperone proteins work inside cells to ensure the proteins assembled there are either properly folded or dispatched for dismantling.

    Regardless of the label, Linda Hendershot, PhD, Genetics and Tumor Cell Biology, said pERp1 is critical for high-level antibody production. She is the study’s senior author. “If the cells don’t have it, they have trouble assembling the disulfide bonds that function like Velcro to help antibodies maintain their proper shapes,” she explained.

    The pERp1 protein is produced at very low levels in white blood cells known as B cells. Working in mice, the investigators showed pERp1 levels in bone marrow and spleen cells jumped dramatically when a foreign marker called an antigen prompted B lymphocytes to become the more specialized, or differentiated, plasma cells that make antibodies. These antibody-producing cells begin life in the bone marrow and end up in the spleen.

    Investigators reported B cells can also be coaxed into making pERp1 through certain activators of a mechanism known as the unfolded protein response (UPR). The UPR signals cells to increase production of chaperone and other folding enzymes essential for proper protein production.

    Antibodies are assembled from three separate amino acid strands, or chains, including two identical strands known as heavy chains and two more known as light chains. Each strand must fold into the proper shape and then come together with a third chain to make the IgM pentameric molecule, whose 21 chains make it the largest circulating human antibody. Ultimately antibodies rely on about 100 disulfide bonds to maintain their correct 3-D shape. With plasma cells churning out about 1,000 antibodies per second, each cell must form about 100,000 disulfide bonds per second.

    Considering the demand, Yuichiro Shimizu, PhD, said it is not surprising plasma cells might have a protein like pERp1 dedicated to helping antibodies fold correctly. Shimizu, the paper’s lead author, just completed a postdoctoral fellowship in Hendershot’s laboratory.

    By manipulating the amount of pERp1 produced in plasma cells, researchers demonstrated that the protein promoted correct folding and stability of the antibody’s two heavy chains.

    Proper antibody folding is a crucial component of antibody function, Hendershot said. “If the antibodies do not form correctly, they might not protect against infection or they might mistakenly react against healthy tissue,” she said.

    Hendershot said the pharmaceutical industry is interested in pERp1 as a possible tool to increase production of antibodies for disease prevention or treatment.

    The research was published in tandem with a paper from investigators led by Ineke Braakman, PhD, Utrecht University, The Netherlands. Hendershot said the two groups found pERp1 independently, but opted to publish their results together with each focusing on a different aspect of the protein.

    Hendershot has a longstanding interest in the system that cells use to ensure the proteins are correctly assembled and folded. The process occurs inside a specialized structure within the cell called the endoplasmic reticulum (ER). She is particularly interested in the role played by members of the BiP family of chaperone proteins. These chaperone proteins ensure proteins fold properly before they are released from the ER and identify defective proteins to be sent to the cell’s internal shredder, a structure called the proteasome.

    More than five years ago, Hendershot’s laboratory launched an effort to better understand how the BiP family of chaperone proteins was organized and functioned in the ER. In a happy coincidence, Laurent Meunier, PhD, then a postdoctoral fellow in Hendershot’s laboratory, began the work in plasma cells, where pERp1 functions. He is a co-author of this study.

    Working with the Hartwell Center for Bioinformatics and Biotechnology, investigators used a technique called mass spectrometry to recognize the new protein in the BiP complex. The researchers used the resulting genetic sequence to trace the new protein to white blood cells. They also used the genetic blueprint to predict how the protein would likely fold and to identify it as a possible new member of a family of enzymes known as protein disulfide isomerases or PDIs.

    Hendershot said the next steps include crystallizing the protein to determine its structure as well as additional research to clarify how pERp1 functions during antibody production.

    The research was supported by the National Institutes of Health and ALSAC.

    January 2010