Tomoya O. Akama

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Fibulin-4 conducts proper elastogenesis via interaction with cross-linking enzyme lysyl oxidase

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.

Carbohydrate-modifying sulfotransferases. Structure, function, and pathophysiology.

Sulfated oligosaccharides play diverse roles in development, differentiation, and homeostasis. For example, heparan sulfate or heparan sulfate-like glycans were shown to play roles in binding growth factors to receptors (1, 2) and adhesion of herpes simplex virus 1 to the cell surface (3). Abolition of sulfation in heparan sulfate synthesis results in neonatal death during mouse development (4) and abnormal development in Drosophila (5, 6). These sulfate groups are formed by sulfotransferases. Sulfotransferases specifically transfer a sulfate group from the sulfate donor substrate, 3 -phosphoadenosine 5 -phosphosulfate (PAPS), to a specific position of a specific carbohydrate residue. Molecular cloning of these sulfotransferases was achieved initially by purifying a given enzyme and cDNA cloning based on the amino acid sequence of the purified enzyme. These pioneering clonings include heparan sulfate N-deacetylase/sulfotransferase (7, 8), chondroitin sulfate GalNAc 6-Osulfotransferase (9), heparan sulfate GlcN 3-O-sulfotransferase (10), and galactosylceramide 3-O-sulfotransferase (11). These studies demonstrate that sulfotransferases also have the same type II membrane topology as other Golgi enzymes such as glycosyltransferases. The crystal structure of an estrogen sulfotransferase revealed the amino acid sequence motifs that correspond to the binding sites for 5 -phosphosulfate and 3 phosphate groups of the donor substrate, PAPS (12). Comparison of the amino acid sequences of these domains shows that they are conserved among the sulfotransferases cloned to date (13). Initial molecular cloning of a sulfotransferase was also achieved by expression cloning using antibodies specific to a given carbohydrate. These studies include cloning of HNK-1 sulfotransferase (HNK-1ST) (14, 15). In one of these studies, it was revealed that there is a conserved amino acid sequence motif ZZRDPXXZ among cloned sulfotransferases, where X and Z denote any amino acid and a hydrophobic amino acid, respectively (14). This motif turned out to correspond to a part of the binding site for the 3 -phosphate group of PAPS. Sitedirected mutagenesis of these amino acids and crystal structure analysis, described above, showed that the arginine 189 and serine 197 in this region are involved in hydrogen bonding to the 3 -phosphate group, whereas the aspartic acid 190 and proline 191 residues reside in the core structure of the 3 phosphate binding site forming a tight turn in the polypeptide (Fig. 1). The lysine 128 residue in the 5 -phosphosulfate binding site may also be involved in binding to an acceptor in which sulfation takes place at the 3 -OH (Fig. 1), because the K128R mutant enzyme showed lower affinity to the acceptor compared with the wild-type enzyme (16). The presence of the weak but discernible similarity among different sulfotransferases suggested the possibility that other sulfotransferases may be identified by their similarity to sulfotransferases already cloned. Indeed, following the cloning of HNK-1ST, two sulfotransferases were cloned based on their similarity to HNK-1ST. Studies on the substrate specificity of these sulfotransferases, however, unexpectedly revealed that these sulfotransferases encode for chondroitin GalNAc 4-Osulfotransferases (Ch4STs) (17, 18). This is rather striking, considering that HNK-1ST and Ch4ST catalyze very different reactions; HNK-1ST adds a sulfate to the 3-position of glucuronic acid, which is in turn attached to the 3-position of galactose in N-acetyllactosamine, whereas Ch4ST adds a sulfate to the 4-position of N-acetylgalactosamine, which is in turn attached to the 4-position of glucuronic acid. The hydroxyl groups in both C-3 of glucuronic acid and C-4 of N-acetylgalactosamine are projected above their respective pyranose rings. It is tempting to speculate that the active sites of HNK-1ST and Ch4ST may approach the acceptor from above the plane of the respective acceptor. These results suggest that seemingly unrelated sulfotransferases could be cloned by their similarity to the probed sulfotransferase (see also Fig. 2). On the other hand, sulfotransferases sharing a similar reaction and acceptor are often related to each other. Following the cloning of Ch4ST, N-acetylgalactosamine 4-O-sulfotransferases (GalNAc4ST-1 and GalNAc4ST-2) that add a sulfate to the 4-position of N-acetylgalactosamine in GalNAc 134GlcNAc3R were thus molecularly cloned based on their similarity (19–21). It has been demonstrated that N-acetylgalactosamine 4-O-sulfation in hormonal glycoproteins such as lutropin is essential for maintaining an effective half-life for the hormonal glycoproteins once they are released into the bloodstream. Unsulfated forms are quickly taken up by galactose-binding lectin in the liver, whereas sialylated forms survive too long in the bloodstream, potentially causing an over-response in target tissues (22). This hypothesis can be tested now by generating mutant mice with defective GalNAc4ST-1 and/or GalNAc4ST-2 by gene targeting.

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase

Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Targeted drug delivery to tumor vasculature by a carbohydrate mimetic peptide

Although numerous carbohydrates play significant roles in mammalian cells, carbohydrate-based drug discovery has not been explored due to the technical difficulty of chemically synthesizing complex carbohydrate structures. Previously, we identified a series of carbohydrate mimetic peptides and found that a 7-mer peptide, designated I-peptide, inhibits hematogenous carbohydrate-dependent cancer cell colonization. During analysis of the endothelial surface receptor for I-peptide, we found that I-peptide bound to annexin 1 (Anxa1). Because Anxa1 is a highly specific tumor vasculature surface marker, we hypothesized that an I-peptide-like peptide could target anticancer drugs to the tumor vasculature. This study identifies IFLLWQR peptide, designated IF7, as homing to tumors. When synthetic IF7 peptide was conjugated to fluorescent Alexa 488 (A488) and injected intravenously into tumor-bearing mice, IF7-A488 targeted tumors within minutes. IF7 conjugated to the potent anticancer drug SN-38 and injected intravenously into nude mice carrying human colon HCT116 tumors efficiently suppressed tumor growth at low dosages with no apparent side effects. These results suggest that IF7 serves as an efficient drug delivery vehicle by targeting Anxa1 expressed on the surface of tumor vasculature. Given its extremely specific tumor-targeting activity, IF7 may represent a clinically relevant vehicle for anticancer drugs.

Latent TGF-β binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-β because TGF-β–free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-β–bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.

Implantation-Dependent Expression of Trophinin by Maternal Fallopian Tube Epithelia during Tubal Pregnancies: Possible Role of Human Chorionic Gonadotrophin on Ectopic Pregnancy

Trophinin, tastin, and bystin have been identified as molecules potentially involved in human embryo implantation. Both trophoblasts and endometrial epithelial cells express trophinin, which mediates apical cell adhesion through homophilic trophinin-trophinin binding. We hypothesized that trophinins function in embryo implantation is unique to humans and investigated the expression of trophinin, tastin, and bystin in ectopic pregnancy, a condition unique to humans. In tubal pregnancies, high levels of all three were found in both trophoblasts and fallopian tubal epithelia. Trophinin expression in maternal cells was particularly high in the area adjacent to the trophoblasts, whereas trophinin was barely detectable in intact fallopian tubes from women with in utero pregnancies or without pregnancies. When explants of intact fallopian tube were incubated with the human chorionic gonadotrophin (hCG), trophinin expression was enhanced in epithelial cells. Since the trophectoderm of the human blastocyst secretes hCG before and after implantation, these results suggest that hCG from the human embryo induces trophinin expression by maternal cells. As both beta-subunit of hCG and trophinin genes have diverged in mammals, the present study suggests a unique role of hCG and trophinin in human embryo implantation, including the pathogenesis of ectopic pregnancy.

Structural and biochemical aspects of keratan sulphate in the cornea

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly-thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.

Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells

Cell surfaces of epithelial cancer are covered by complex carbohydrates, whose structures function in malignancy and metastasis. However, the mechanism underlying carbohydrate-dependent cancer metastasis has not been defined. Previously, we identified a carbohydrate-mimicry peptide designated I-peptide, which inhibits carbohydrate-dependent lung colonization of sialyl Lewis X-expressing B16-FTIII-M cells in E/P-selectin doubly-deficient mice. We hypothesized that lung endothelial cells express an unknown carbohydrate receptor, designated as I-peptide receptor (IPR), responsible for lung colonization of B16-FTIII-M cells. Here, we visualized IPR by in vivo biotinylation, which revealed that the major IPR is a group of 35-kDa proteins. IPR proteins isolated by I-peptide affinity chromatography were identified by proteomics as Ser/Arg-rich alternative pre-mRNA splicing factors or Sfrs1, Sfrs2, Sfrs5, and Sfrs7 gene products. Bacterially expressed Sfrs1 protein bound to B16-FTIII-M cells but not to parental B16 cells. Recombinant Sfrs1 protein bound to a series of fucosylated oligosaccharides in glycan array and plate-binding assays. When anti-Sfrs antibodies were injected intravenously into mice, antibodies labeled a subset of lung capillaries. Anti-Sfrs antibodies inhibited homing of I-peptide-displaying phage to the lung colonization of B16-FTIII-M cells in vivo in the mouse. These results strongly suggest that Sfrs proteins are responsible for fucosylated carbohydrate-dependent lung metastasis of epithelial cancers.

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.