Mucin-type O-glycosylation is critical for vascular integrity

Abstract

Vascular endothelial cells, in addition to many other mammalian cell types, express proteins that are highly modified with mucin-type O-glycosylation, a specific type of glycosylation that begins with the addition of an N-acetylgalactosamine moiety to serine or threonine residues within the peptide backbone. Recently, it has become evident that O-glycosylation governs the separation of blood and lymphatic vessels throughout life and plays a critical role in maintaining vascular integrity in specific tissues such as the brain and lymph node. This mini-review seeks to highlight some of these recent advances regarding in vivo functions of mucin-type O-glycans.

Introduction

Glycosylation is a common posttranslational modification of proteins and refers to the addition of a multitude of monosaccharides (glycans) to peptides (Kornfeld and Kornfeld 1985; Varki et al. 2009.). These complex modifications allow for greater functional diversity of proteins. O-Glycosylation is a common form of protein posttranslational modification, which is increasingly appreciated as having a wide and yet largely unexplored spectrum of biological functions. There are several types of O-glycans. This review focuses on mucin-type O-glycans (also known as O-N-acetylgalactosamine glycans or simply O-glycans for this review) that are covalently α-linked via a GalNAc moiety to the hydroxyl (–OH) group of serine or threonine residues within a peptide backbone (Varki et al. 2009; Tran and Ten Hagen 2013). O-Glycans are well characterized for their roles in mediating leukocyte trafficking during inflammation and thrombosis (McEver 2001). Recently, it has become evident that mucin-type O-glycosylation plays a critical role in maintaining vascular integrity in vivo. This review seeks to highlight some of the recent advances.

Mucin-type O-glycosylation

Mucin-type O-glycosylation is an essential form of protein posttranslational modification. It is initiated by a class of glycosyltransferases, polypeptidyl N-acetylgalactosaminetransferases, which attach a GalNAc from a uridine diphosphate (UDP)-GalNAc to serine or threonine residues on a peptide backbone creating the Tn antigen (Varki et al. 2009). Two major forms of mucin-type O-glycans exist: core 1- and core 3-derived (Figure 1). Core 3-derived O-glycans are primarily restricted to the intestinal mucosa and salivary glands (Iwai et al. 2002; An et al. 2007), whereas core 1-derived O-glycans are widely expressed on many cells types, including endothelial cells, neural cells, stromal cells and hematopoietic cells (Fu et al. 2008; Xia et al. 2004). Core 1-derived O-glycans are formed through the action of a singular enzyme, core 1 β1,3-galactosyltransferase (T-synthase) (Ju et al. 2002). Proper folding and ultimately, the function of T-synthase requires its endoplasmic reticulum-located molecular chaperone, Cosmc (Ju and Cummings 2002). T-synthase adds a galactose (Gal) molecule from UDP-Gal to the Tn antigen to form the T antigen, the basis for core 1-derived O-glycans (Varki et al. 2009) (Figure 1). Core 1-derived O-glycans are highly expressed by endothelial cells, where they play important roles in leukocyte adhesion and endothelial stability (McEver and Cummings 1997; Xia et al. 2004; Yago et al. 2010). Furthermore, it is becoming increasingly apparent that O-glycans and O-glycoproteins play important roles during the initial establishment of the vascular compartments and the maintenance of their integrity (Xia et al. 2004; Fu et al. 2008; Herzog et al. 2013).

Fig. 1.

The biosynthesis of core 1 and core 3 O-glycans. Arrowheads indicate further branching to form core 1- or 3-derived O-glycans.

O-glycans and O-glycoproteins in blood vascular development

The formation of vascular networks is critical for proper mammalian development. Within the developing embryo, two distinct vascular networks take shape: the blood and lymphatic vessel networks. Each network is unique and serves distinct functions; however, endothelial cells, which form conduits for carrying cells, small molecules and waste products, line them both. During development, de novo blood vessel formation occurs through a process called vasculogenesis whereby endothelial cells differentiate from precursor cells (Carmeliet 2000). Further formation of vascular networks occurs through a process known as angiogenesis, which involves endothelial cell division and migration. Ultimately, vascular branching and pruning gives rise to the mature blood vascular networks observed in adults. As embryonic development progresses, an additional vessel type, lymphatics, differentiates from venous precursors (Oliver 2004). While these lymphatic vessels ultimately develop a distinct vascular system through lymphangiogenesis, functional connections exist between lymphatics and veins that allow lymphatics to return their contents to the systemic circulation for use or clearance by the body. These connections maintain unidirectional flow through lymphatics such that blood does not typically flow through lymphatic vessels. Further phenotypic differentiation occurs along the vascular tree so that large transport vessels, capillaries and postcapillary venules are able to perform specialized functions. Such is the case with specialized postcapillary venules called high endothelial venules (HEVs) in lymph nodes, which exhibit specific molecules that facilitate lymphocyte rolling and transmigration for immune surveillance (McEver and Cummings 1997; Girard et al. 2012).

Endothelial cells express high levels of O-glycans. However, the functional role of O-glycosylation in vascular development and stability are only beginning to be appreciated. An unexpected role of core 1 O-glycosylation was revealed by genetic deletion of T-synthase in mice, which led to the identification of a critical role for O-glycans in vascular integrity (Xia et al. 2004). Mice lacking T-synthase (T-syn^−/−) form defective vascular networks in the central nervous system and died from massive brain hemorrhage at embryonic day 13 or 14. Vessels from T-syn^−/− mice display abnormal lumens and endothelial cells exhibit abnormal sprouting and decreased pericyte coverage, suggesting that O-glycans play a critical role in endothelial–endothelial cell interactions as well as in cross-talk between endothelial and supporting cells (Xia et al. 2004). However, further investigation demonstrated that endothelial and hematopoietic cell-specific deletion of T-synthase in mice (EHC T-syn^−/−) was not sufficient to induce hemorrhage in the central nervous system as observed in T-syn^−/− mice, suggesting that cell types in addition to O-glycan expressing blood/endothelial cells are required for maintaining vascular integrity in the brain during development (Fu et al. 2008). Furthermore, mice with inducible deletion of T-synthase after birth do not exhibit the spontaneous brain bleeding phenotype, which suggests that O-glycan requirement for vascular integrity in the central nervous system is temporal in nature (Fu et al. 2008). The brain vasculature is unique. Early in development, it provides a specialized environment in which the developing brain is protected from noxious stimuli and later develops a fully functional blood–brain barrier. As the embryo is developing, the perineural plexus forms through vasculogenesis. These nascent vessels invade the brain parenchyma through angiogenesis and form a primitive vascular network (Ballabh 2010). Studies from T-syn^−/− mice suggest that O-glycans play a role in normal association of pericytes as well as stabilization of the nascent vasculature (Xia et al. 2004). Additional studies are required to delineate which O-glycoprotein(s) on which cell(s) are critical in maintaining cerebrovascular integrity during development.

Blood and lymphatic vessel separation requires O-glycans

Although EHC T-syn^−/− mice do not develop spontaneous hemorrhage in the brain, they develop blood-filled lymphatic vessels early during development (Fu et al. 2008). Their lymphatic vessels fail to functionally separate from blood vessels and exhibit dilated, blood-filled lymphatic vessels (Fu et al. 2008). Similar phenotypes were also found in mice that lack the T-synthase chaperone protein, Cosmc, in endothelial and hematopoietic cells (Wang et al. 2012). Although EHC T-syn^−/− mice lack O-glycans on endothelial and hematopoietic cells, it is endothelial cell O-glycans that contribute to the separation of blood and lymphatic vessels as mice with O-glycan deficiency in hematopoietic cells alone develop normal blood-lymphatic separation (Fu et al. 2008). Further analysis of EHC T-syn^−/− endothelial cells led to the first demonstration that podoplanin, an O-glycoprotein (Figure 2), requires O-glycosylation for normal expression/function on lymphatic endothelial cells. Indeed, similar to EHC T-syn^−/− mice, mice lacking podoplanin develop blood-filled lymphatics vessels due to a failure to functionally separate from blood vessels during development (Fu et al. 2008).

Fig. 2.

Interaction between the O-glycoprotein, podoplanin (PDPN) and its platelet receptor, CLEC-2. Normal expression of podoplanin at the cell surface requires the activity of T-synthase (T-syn) to properly modify the core protein structure with core 1-derived O-glycans. Podoplanin on lymphatic endothelial cells (LECs) or fibroblastic reticular cells (FRCs) activates platelet CLEC-2, which induces downstream signaling events including activation of the tyrosine kinase, Syk and ultimately, platelet activation and/or aggregation. This mechanism is important in both ensuring and maintaining separation of blood and lymphatic vessels as well as stabilizing HEV integrity within lymph nodes.

The only known physiological receptor for podoplanin is the C-type lectin-like receptor 2 (CLEC-2) that is primarily expressed on platelets. O-Glycans located on podoplanin are essential for interacting with CLEC-2 in vitro (Kaneko et al. 2004; Kaneko et al. 2007). During development, properly O-glycosylated podoplanin is expressed at the surface of lymphatic endothelial cells where it interacts with CLEC-2 on platelets. Podoplanin engagement of platelet CLEC-2 initiates the intracellular recruitment of signaling molecules Syk, SLP-76 and PLCγ2 resulting in activation and aggregation of platelets (Abtahian et al. 2003; Suzuki-Inoue et al. 2007; Ichise et al. 2009; Bertozzi et al. 2010). Platelet activation via podoplanin-CLEC-2 interaction leads to the initial functional separation of blood and lymphatic vessels (Figure 2) (Fu et al. 2008; Uhrin et al. 2010; Hess et al. 2014).

In addition, postnatal loss of O-glycans causes blood-filled lymphatic vessels in mice, indicating a continued requirement for O-glycans in maintaining the integrity and functional separation of the blood and lymphatic systems (Fu et al. 2008). Indeed, our recent studies demonstrate that following the initial separation of blood and lymphatic vessels, the O-glycan-dependent podoplanin-CLEC-2 interaction maintains the functional separation and prevents retrograde flow of blood into lymphatics by maintaining lymphovenous valve integrity (Hess et al. 2014).

O-Glycans in maintaining HEV integrity in the lymph node

In addition to developing blood-filled lymphatics, mice lacking T-synthase or podoplanin postnatally develop bleeding within the lymph nodes (Herzog et al. 2013) (Figure 3). Lymph nodes are essential sites for immune responses (Drayton et al. 2006). Circulating naive lymphocytes constantly enter lymph nodes in a process commonly called lymphocyte homing (Kunkel and Butcher 2002; von Andrian and Mempel 2003; Rosen 2004). There they encounter antigens or antigen-presenting cells delivered by afferent lymphatic vessels from surrounding tissues. Lymphocyte homing occurs exclusively through the vessel wall of the HEVs, which are specialized vessels composed of tall, plump endothelial cells. HEVs express specific adhesion molecules that enable lymphocyte rolling, arrest and transmigration through the vessel wall. HEVs have particular “spot-weld” cell–cell junctions called adherens junctions, which are primarily composed of VE-cadherin (Dejana 1996; Dejana et al. 2009; Girard et al. 2012). Importantly, HEVs are surrounded by specialized stromal cells called fibroblastic reticular cells, which express the O-glycoprotein, podoplanin (Schacht et al. 2003; Bajenoff et al. 2006; Chyou et al. 2008; Fu et al. 2008). During an immune response, lymphocyte trafficking through HEVs can increase by >10-fold, but how HEVs handle such flux across their junctions while retaining their vascular integrity is only now coming to light. Recent studies show that mice with postnatal inducible deletion or selective deletion of podoplanin in fibroblastic reticular cells exhibit spontaneous bleeding in the mucosal lymph nodes, a characteristic that also occurred in mice with platelet-specific deficiency of CLEC-2 (Herzog et al. 2013). Further investigation of mice with postnatal podoplanin deficiency revealed that the HEVs within the lymph nodes exhibit decreased vascular integrity such that intravenously injected, labeled red blood cells leaked from HEVs in podoplanin-deficient mice (Herzog et al. 2013). We found that platelets are able to access the perivascular space around HEVs in a lymphocyte transmigration-dependent manner. Once at the extralumenal surface of HEVs, platelet CLEC-2 is activated by extravascular podoplanin on fibroblastic reticular cells (Figure 2). Podoplanin activation of platelet CLEC-2 induces the release of sphingosine 1-phosphate (S1P), a bioactive lipid, from platelets, which in turn binds to its receptor, S1PR1, on HEVs. Activation of S1PR1 increases HEV integrity by promoting VE-cadherin reassembly at the endothelial adherens junctions effectively sealing the functional leakage caused by migrating lymphocytes (Herzog et al. 2013). This study highlights the essential role of podoplanin, a mucin-type O-glycoprotein, in maintaining HEV integrity by interacting with CLEC-2 on platelets to trigger the release of platelet S1P in the perivenular space that preserves VE-cadherin expression on HEVs. Thus, cross-talk between fibroblastic reticular cells, platelets and HEVs is essential to maintain HEV integrity in situations of increased lymphocyte trafficking. These findings may lead to approaches for regulating lymph node homeostasis under conditions of increased HEV proliferation such as chronic inflammation.

Fig. 3.

Lymph node bleeding following postnatal loss of T-syn or podoplanin (PDPN). Gross images of cervical lymph nodes from wild-type mice and mice with postnatal deletion of T-syn or PDPN. Scale bar is 1 mm.

Future prospects

O-Glycosylation of podoplanin governs the initial separation of blood and lymphatic vessels during development, and continues to maintain blood and lymphatic separation throughout life. However, how O-glycans regulates functional expression of podoplanin on lymphatic endothelial cells to maintain functional separation of blood and lymphatic vessels remains unclear and warrants further investigation. Studies from T-syn^−/− mice suggest that O-glycans play a role in normal association of pericytes as well as stabilization of the nascent vasculature in the brain. In particular, areas within the subventricular zone appeared especially susceptible to deficiencies in O-glycosylation (Xia et al. 2004). However, questions remain regarding how O-glycosylation regulates vascular development in the central nervous system because blood vascular endothelial cells do not express podoplanin. In addition, whether O-glycosylation protects vessel integrity in additional vascular beds or during rapid vessel remodeling such as that occurs during wound healing or in tumor development is unknown. Furthermore, while published studies have revealed major O-glycoproteins such as podoplanin in vascular development and integrity, it is possible that other O-glycoproteins may also contribute to the integrity and development of blood and lymphatic vessels. The recent development of new cell type-specific strains of O-glycan-deficient mice will continue to enable the investigation of these questions as well as identify novel O-glycoproteins that contribute to previously observed phenotypes.

Abbreviations

CLEC-2, C-type lectin-like receptor 2; FRCs, fibroblastic reticular cells; Gal, galactose; GalNAc, N-acetylgalactosamine; HEVs, high endothelial venules; LECs, lymphatic endothelial cells; S1P, sphingosine-1-phosphate; UDP, uridine diphospho

Conflict of interest statement

None declared.

Funding

Work was supported by grants from the National Institute of Health (GM103441, HL085607) and the American Heart Association (11SDG7410022).