Any insufficiency of hematopoiesis, from iatrogenic causes secondary to cancer chemoradiotherapy, from exposure to toxins (e.g., benzene), or from unknown etiology, poses significant risks of mortality, mostly due to bleeding and infections. All of these conditions may be ameliorated by more rapid and effective reestablishment of hematopoietic function. There is increasing evidence that sialyl and fucosyl derivatives of lactosaminyl glycans, such as sialyl Lewis X structures, mediate adhesive interactions critical to hematopoietic stem and progenitor cell homeostasis. However, the glycan structural profiles of early hematopoietic cells, the glycosylation network governing their stage- and lineage-specific expression, and their precise participation in hematopoietic process(es), are largely unknown. In this proposal, we will focus on the biosynthetic pathways governing sialofucosylations that modify lactosamine structures. We will extend these studies to examine how glycosyltransferases influence commitment into myeloid and megakaryocyte lineages. By implementing a three-tiered approach of glycogene expression query, enzymatic activity profiling, and glycan structural analysis, we wiil elucidate hematopoietic stem and progenitor cell surface glycan structures and identify the key glycan-modifying enzymatic activities occupying the biosynthetic checkpoints in the production of these structures. In addition to characterizing the conventional ER/Golgi-based network of glycosyltransferases, we will explore a novel pathway of glycan synthesis uncovered by recent studies in our lab. The canonical view of glycan synthesis holds that glycosylation events occur only within the same cell that expresses the cognate glycosyltransferase(s). However, we have evidence for a novel alternate pathway of glycosylation whereby hematopoietic cell surface glycans can be remodeled extracellularly, by "extrinsic" enzymes originating from distal sources, and point to the idea of extrinsic enzymes as factors in regulating hematopoiesis. The extent ofthe involvement of the extrinsic enzymes in generating hematopoietic cell surface glycans will be evaluated by forced expression of circulatory recombinant enzymes, and also by construction of bone marrow chimeras using donor hematopoietic cells with specific glycosyltransferase defects into wild-type recipients, and vice versa. The biologic roles of specific glycans, whether they are of intrinsic or extrinsic construction, will be assessed by ex vivo adhesion and clonogenic assays and by in vivo homing, retention, repopulation, and trafficking parameters. A mathematical modeling framework will be developed to enhance our understanding of dynamic glycosylation pathways, and therefore how they can be manipulated for therapeutic benefit. We anticipate the results of our studies will yield novel strategies for glycan engineering of hematopoietic cell surfaces towards modification of their biologic behavior.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL107146-03
Application #
8477245
Study Section
Special Emphasis Panel (ZHL1-CSR-H)
Project Start
Project End
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
3
Fiscal Year
2013
Total Cost
$605,864
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
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