Jamey D. Marth

A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA

Protein-tyrosine kinases are implicated in the control of normal and neoplastic cell growth. We have used molecular cloning strategies to characterize a lymphocyte-specific protein-tyrosine kinase gene distinct from but closely related to src and yes. This gene, encoded by a genetic locus defined here as lskT, is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Thus alterations in the structure or expression of this protein-tyrosine kinase gene may in some cases mediate neoplastic transformation. In addition, transcription of the normal lskT gene is restricted to cells of lymphoid origin. We infer that the lskT-encoded protein-tyrosine kinase may aid in transducing proliferative or differentiative signals unique to lymphocytes.

The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis.

T lymphocyte activation evokes distinct changes in cell surface O-glycans. CD8+ T cells undergo an elimination of sialic acid on core 1 O-glycans and an induction of core 2 O-glycans until either apoptotic death or differentiation into memory cells. We find that the ST3Gal-I sialyltransferase is required for core 1 O-glycan sialylation and its deficiency induces core 2 O-glycan biosynthesis. Apoptosis ensues with the loss of peripheral CD8+ T cells in the absence of immune stimulation. Cell surface ligation of the ST3Gal-I substrate CD43 recapitulates this phenotype by a caspase 3-independent mechanism. Control of core 1 O-glycan sialylation in T lymphocytes by ST3Gal-I comprises a homeostatic mechanism that eliminates CD8+ T cells by apoptosis while facilitating the production of viable CD8+ memory T cells.

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Brca1 required for T cell lineage development but not TCR loci rearrangement.

Brca1 (breast cancer1, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell–specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1−/− mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53−/− backgrounds, completely restored survival and development of Brca1−/− thymocytes; peripheral T cell numbers were not totally restored in Brca1−/− p53−/− mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1−/− thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

Dendritic Cell-Dependent Inhibition of B Cell Proliferation Requires CD22

Recent studies have shown that dendritic cells (DCs) regulate B cell functions. In this study, we report that bone marrow (BM)-derived immature DCs, but not mature DCs, can inhibit BCR-induced proliferation of B cells in a contact-dependent manner. This inhibition is overcome by treatment with BAFF and is dependent on the BCR coreceptor CD22; however, it is not dependent on expression of the CD22 glycan ligand(s) produced by ST6Gal-I sialyltransferase. We found that a second CD22 ligand (CD22L) is expressed on CD11c+ splenic and BM-derived DCs, which does not contain ST6Gal-I-generated sialic acids and which, unlike the B cell-associated CD22L, is resistant to neuraminidase treatment and sodium metaperiodate oxidation. Examination of splenic and BM B cell subsets in CD22 and ST6Gal-I knockout mice revealed that ST6Gal-I-generated B cell CD22L plays a role in splenic B cell development, whereas the maintenance of long-lived mature BM B cells depends only on CD22 and not on α2,6-sialic acids produced by ST6Gal-I. We propose that the two distinct CD22L have different functions. The α2,6-sialic acid-containing glycoprotein is important for splenic B cell subset development, whereas the DC-associated ST6Gal-I-independent CD22L may be required for the maintenance of long-lived mature B cells in the BM.

High Sensitivity O-glycomic Analysis of Mice Deficient in Core 2 β1,6-N-acetylglucosaminyltransferases

Core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1-3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500xa0Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids.

Differential Requirements for Core2 Glucosaminyltransferase for Endothelial L-Selectin Ligand Function In Vivo

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 β1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-α-treated cremaster muscles and in Peyer’s patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2−/−) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2−/− mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer’s patch HEV was not significantly different between core2−/− and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2−/− mice, but significant rolling in controls. In Peyer’s patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2−/− mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2−/− and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer’s patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.

A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins

The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease.

Computational modeling of glucose transport in pancreatic β-cells identifies metabolic thresholds and therapeutic targets in diabetes.

Pancreatic β-cell dysfunction is a diagnostic criterion of Type 2 diabetes and includes defects in glucose transport and insulin secretion. In healthy individuals, β-cells maintain plasma glucose concentrations within a narrow range in concert with insulin action among multiple tissues. Postprandial elevations in blood glucose facilitate glucose uptake into β-cells by diffusion through glucose transporters residing at the plasma membrane. Glucose transport is essential for glycolysis and glucose-stimulated insulin secretion. In human Type 2 diabetes and in the mouse model of obesity-associated diabetes, a marked deficiency of β-cell glucose transporters and glucose uptake occurs with the loss of glucose-stimulated insulin secretion. Recent studies have shown that the preservation of glucose transport in β-cells maintains normal insulin secretion and blocks the development of obesity-associated diabetes. To further elucidate the underlying mechanisms, we have constructed a computational model of human β-cell glucose transport in health and in Type 2 diabetes, and present a systems analysis based on experimental results from human and animal studies. Our findings identify a metabolic threshold or “tipping point” whereby diminished glucose transport across the plasma membrane of β-cells limits intracellular glucose-6-phosphate production by glucokinase. This metabolic threshold is crossed in Type 2 diabetes and results in β-cell dysfunction including the loss of glucose stimulated insulin secretion. Our model further discriminates among molecular control points in this pathway wherein maximal therapeutic intervention is achieved.

Deficiency of the Sialyltransferase St3Gal4 Reduces Ccl5-Mediated Myeloid Cell Recruitment and Arrest

Rationale: Sialylation by &agr;2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. Objective: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of &agr;2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis. Methods and Results: St3Gal4-deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-&agr;–stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4−/− mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe−/− mice on high-fat diet. Conclusions: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.