Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs.

HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1.

BACKGROUND: Heparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin. RESULTS: Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities. CONCLUSION: Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer.

Sulf-2 is an endosulfatase with activity against glucosamine-6-sulfate modifications within subregions of intact heparin. The enzyme has the potential to modify the sulfation status of extracellular heparan sulfate proteoglycan (HSPG) glycosaminoglycan chains and thereby to regulate interactions with HSPG-binding proteins. In the present investigation, data mining from published studies was employed to establish Sulf-2 mRNA upregulation in human breast cancer. We further found that cultured breast carcinoma cells expressed Sulf-2 mRNA and released enzymatically active proteins into conditioned medium. In two mouse models of mammary carcinoma, Sulf-2 mRNA was upregulated in comparison to its expression in normal mammary gland. Although mRNA was present in normal tissues, Sulf-2 protein was undetectable; it was, however, detected in some premalignant lesions and in tumors. The protein was localized to the epithelial cells of the tumors. In support of the possible mechanistic relevance of Sulf-2 upregulation in tumors, purified recombinant Sulf-2 promoted angiogenesis in the chick chorioallantoic membrane assay.

Detection of a sulfotransferase (HEC-GlcNAc6ST) in high endothelial venules of lymph nodes and in high endothelial venule-like vessels within ectopic lymphoid aggregates: relationship to the MECA-79 epitope.

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules (HEVs) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte homing. The MECA-79 monoclonal antibody reports HEV-expressed ligands for L-selectin by recognizing a critical sulfation-dependent determinant on these ligands. HEC-GlcNAc6ST, a HEV-localized sulfotransferase, is essential for the elaboration of functional ligands within lymph nodes, as well as the generation of the MECA-79 epitope. Here, we use an antibody against murine HEC-GlcNAc6ST to study its expression in relationship to the MECA-79 epitope. In lymph nodes, the enzyme is expressed in the Golgi apparatus of high endothelial cells, in close correspondence with luminal staining by MECA-79. In lymph node HEVs of HEC-GlcNAc6ST-null mice, luminal staining by MECA-79 is almost abolished, whereas abluminal staining persists although reduced in intensity. HEV-like vessels in several examples of inflammation-associated lymphoid neogenesis, including nonobese diabetic mice, also exhibit concomitant expression of the sulfotransferase and luminal MECA-79 reactivity. The correlation extends to ectopic lymphoid aggregates within the pancreas of RIP-BLC mice, in which CXCL13 is expressed in islets. Analysis of the progeny of RIP-BLC by HEC-GlcNAc6ST-null mice establishes that the enzyme is responsible for the MECA-79 defined luminal ligands.

Lymphocyte-HEV interactions in lymph nodes of a sulfotransferase-deficient mouse.

The interaction of L-selectin expressed on lymphocytes with sulfated sialomucin ligands such as CD34 and GlyCAM-1 on high endothelial venules (HEV) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte recruitment. HEC-GlcNAc6ST-deficient mice lack an HEV-restricted sulfotransferase with selectivity for the C-6 position of N-acetylglucosamine (GlcNAc). HEC-GlcNAc6ST-/- animals exhibit faster lymphocyte rolling and reduced lymphocyte sticking in HEV, accounting for the diminished lymphocyte homing. Isolated CD34 and GlyCAM-1 from HEC-GlcNAc6ST-/- animals incorporate approximately 70% less sulfate than ligands from wild-type animals. Furthermore, these ligands exhibit a comparable reduction of the epitope recognized by MECA79, a function-blocking antibody that reacts with L-selectin ligands in a GlcNAc-6-sulfate-dependent manner. Whereas MECA79 dramatically inhibits lymphocyte rolling and homing to lymph nodes in wild-type mice, it has no effect on HEC-GlcNAc6ST-/- mice. In contrast, in vitro rolling on purified GlyCAM-1 from HEC-GlcNAc6ST-/- mice, although greatly diminished compared with that on the wild-type ligand, is inhibited by MECA79. Our results demonstrate that HEC-GlcNAc6ST contributes predominantly, but not exclusively, to the sulfation of HEV ligands for L-selectin and that alternative, non-MECA79-reactive ligands are present in the absence of HEC-GlcNAc6ST.

Activities and expression pattern of the carbohydrate sulfotransferase GlcNAc6ST-3 (I-GlcNAc6ST): functional implications.

In recent years, a family of five GlcNAc-6-O-sulfotransferases, called the GlcNAc6STs, has been molecularly cloned. One of these, GlcNAc6ST-2 (originally named HEC-GlcNAc6ST or LSST), shows a very restricted expression at the mRNA level in high endothelial cells (HECs) of lymph nodes high endothelial venules (HEVs). This enzyme has been shown to be involved in elaborating the 6-sulfo sLex structure on a set of mucin-like acceptors within HECs, thus providing a critical recognition determinant for L-selectin during the process of lymphocyte homing to lymph nodes. Limited information has been available about the closely related sulfotransferase known as GlcNAc6ST-3 (I-GlcNAc6ST). Here, employing transfection experiments with a series of glycoprotein acceptors, we report that this sulfotransferase has a marked preference for sulfating O-linked sugars of mucin-type acceptors, whereas other sulfotransferases in the family (GlcNAc6ST-1, GlcNAc6ST-2) and a Gal-6-O-sulfotransferase exhibit strong activity on both mucin-type acceptors and glycoproteins with predominantly N-linked chains. PCR analysis of cDNAs derived from a panel of tissues and purified cell populations confirms the strong expression of GlcNAc6ST-3 in gut-associated tissues and extends the expression to include lymphocytes. In contrast to GlcNAc6ST-2, GlcNAc6ST-3 transcripts are present minimally, if at all, in HECs; moreover, this enzyme is not able to generate the 6-sulfo sLex epitope in transfected cells. These latter findings argue that GlcNAc6ST-3 is not involved in generating HEV-expressed ligands for L-selectin.