Establishment of a human ovarian clear cell carcinoma cell line mutant in PIK3CB but not PIK3CA.

A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient's tumour tissue. Notably, MTC-22 cells, and the patient's carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations-one in ARID1A, which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase-were seen in the patient's tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy.

Structural Elucidation and Prognostic Relevance of 297-11A-Sulfated Glycans in Ovarian Carcinoma.

Ovarian carcinoma is usually diagnosed at an advanced stage with peritoneal dissemination and/or lymph node metastasis, and the prognosis for such advanced carcinoma is very poor. Therefore, new biomarkers to predict patient prognosis are needed. Miyamoto et al. previously showed that keratan sulfate (KS) detected by the 5D4 monoclonal antibody was expressed in ovarian carcinoma. However, the detailed structure of such KS was not determined, and the biological significance of this finding remained to be clarified. We previously generated the 297-11A monoclonal antibody, which recognizes galactose (Gal)-6-O-sulfated N-acetyllactosamine (LacNAc) located at the nonreducing terminus. Because the 297-11A epitope overlaps with that of 5D4, here we chose to use the 297-11A antibody as a tool to analyze KS and related structures. We conducted immunohistochemical analysis of 98 ovarian carcinoma cases with 297-11A antibody combined with a series of glycosidases and performed mass spectrometry analysis of the human serous ovarian carcinoma cell line OVCAR-3 to deduce the glycan structure of 297-11A-sulfated glycans. We also performed western blot analysis to assess a potential association of 297-11A-sulfated glycans with the mucin core protein mucin 16 (MUC16; also known as cancer antigen 125 (CA125)). Finally, we examined the relationship between 297-11A expression and patient prognosis. Consequently, 297-11A-sulfated glycans were primarily expressed in serous and endometrioid carcinomas and poorly expressed in mucinous and clear cell carcinomas. We reveal that structurally, 297-11A-sulfated glycans expressed in ovarian carcinoma are O-glycans carrying partially sialylated, Gal-6-O-sulfated LacNAc and that these glycans are likely displayed on MUC16 mucin core proteins. Of clinical importance is that expression of 297-11A-sulfated glycans correlated with shorter progression-free survival in patients. Thus, 297-11A-sulfated glycans may serve as a predictor of ovarian carcinoma recurrence.

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.

Expression of low-sulfated keratan sulfate in non-mucinous ovarian carcinoma.

Keratan sulfate glycosaminoglycan is composed of repeating N-acetyllactosamine (LacNAc) disaccharide units consisting of galactose (Gal) and N-acetylglucosamine (GlcNAc), both often 6-O-sulfated. Sulfate contents of keratan sulfate are heterogeneous depending upon the origins. In this study, keratan sulfate is classified as either highly sulfated (in which both GlcNAc and Gal residues are 6-O-sulfated) or low-sulfated (in which only GlcNAc residues are 6-O-sulfated). It is reported that highly sulfated keratan sulfate detected by the 5D4 monoclonal antibody is preferentially expressed in normal epithelial cells lining the female genital tract and in their neoplastic counterparts; however, expression of low-sulfated keratan sulfate in either has not been characterized. In the present study, we generated the 294-1B1 monoclonal antibody, which selectively recognizes low-sulfated keratan sulfate, and performed precise glycan analysis of sulfated glycans expressed on human serous ovarian carcinoma OVCAR-3 cells. We found that OVCAR-3 cells do not express highly sulfated keratan sulfate but rather express low-sulfated form, which was heterogeneous in 294-1B1 reactivity. Comparison of mass spectrometry spectra of sulfated glycans in 294-1B1-positive versus -negative OVCAR-3 cells indicated that the 294-1B1 epitope is likely at least 2, and possibly 3 or more, tandem GlcNAc-6-O-sulfated LacNAc units. Then, using the 294-1B1 antibody, we performed quantitative immunohistochemical analysis of 40 specimens from patients with ovarian cancer, consisting of 10 each of serous, endometrioid, clear cell, and mucinous carcinomas, and found that among them low-sulfated keratan sulfate was widely expressed in all but mucinous ovarian carcinoma.

Expression and Clinical Significance of Spi-B in B-cell Acute Lymphoblastic Leukemia.

Spi-B, a member of the E26 transformation-specific (ETS) family of transcription factors, plays an important role in B cell differentiation. Spi-B also functions in development of diffuse large B-cell lymphoma; thus, we hypothesized that it may participate in leukemogenesis of B-cell acute lymphoblastic leukemia (B-ALL). To test this hypothesis, we first generated an anti-Spi-B monoclonal antibody that recognized Spi-B on formalin-fixed, paraffin-embedded tissue sections. This antibody, designated S28-5, selectively stained B cell nuclei at the pre-plasma cell stage (including centrocytes and centroblasts in germinal centers) and nuclei of plasmacytoid dendritic cells, but not fully differentiated plasma cells, T cells, macrophages, or follicular dendritic cells. Employing S28-5, we then performed immunohistochemical staining of bone marrow aspiration biopsy specimens obtained from B-ALL patients (n=62). Cases that showed stronger nuclear S28-5 signals than T-cell ALL were scored positive. In 26 (42%) of 62 specimens, leukemic cells showed nuclear Spi-B expression, and positivity was associated with patient age at diagnosis, and serum uric acid and creatinine levels. Moreover, Spi-B-positive patients demonstrated significantly shorter overall survival than did Spi-B-negative patients. These results suggest that Spi-B expression may serve as a prognostic indicator of B-ALL.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

Efficacy of Nanofiber Sheets Incorporating Lenvatinib in a Hepatocellular Carcinoma Xenograft Model.

Lenvatinib has a high response rate in unresectable advanced hepatocellular carcinoma (HCC). In this study, we investigated whether lenvatinib-incorporating poly(ε-caprolactone) sheets (lenvatinib sheets) as a drug delivery system (DDS) exerted antitumor effects in a murine HCC model. The lenvatinib sheets were designed for sustained release of approximately 1 mg lenvatinib for 14 days. For 14 days, 1 mg lenvatinib was orally administered to mice. Then, we compared the antitumor effects of lenvatinib sheets with those of oral lenvatinib. The tumor volume, body weight, and serum lenvatinib level were measured for 14 days. A peritoneal dissemination model was established to examine the survival prolongation effect of the lenvatinib sheets. Tumor growth was significantly inhibited in the lenvatinib sheet group compared with that in the no treatment and oral groups. The antitumor effect was significantly higher in the lenvatinib sheet group. Regardless of the insertion site, the serum lenvatinib levels were maintained and showed similar antitumor effects. The mitotic index was significantly inhibited in the lenvatinib sheet group compared with that in the control group. Furthermore, lenvatinib sheets improved the 30-day survival. Lenvatinib sheets showed sufficient antitumor effects and may serve as an effective novel DDS for advanced HCC.

Vascular E-selectin Expression Detected in Formalin-fixed, Paraffin-embedded Sections With an E-selectin Monoclonal Antibody Correlates With Ulcerative Colitis Activity.

It is widely accepted that E-selectin, an inducible endothelial cell adhesion molecule, plays a critical role in the initial step of neutrophil recruitment to sites of acute inflammation. However, immunohistological analysis of E-selectin has been hampered by lack of E-selectin-specific monoclonal antibodies that can stain formalin-fixed, paraffin-embedded (FFPE) tissue sections. Here, we employed E-selectin•IgM (a soluble form of E-selectin) as immunogen, and then, after negative selection with L-selectin•IgM and P-selectin•IgM and screening of FFPE sections of both COS-1 cells overexpressing E-selectin and acute appendicitis tissues, we successfully generated an E-selectin-specific monoclonal antibody capable of staining FFPE tissue sections. We used this antibody, designated U12-12, to perform quantitative immunohistological analysis of 390 colonic mucosal biopsy specimens representing ulcerative colitis. We found that the higher the histological disease activity, the greater the number of vessels expressing E-selectin, an observation consistent with previous analyses of frozen tissue sections. Furthermore, in active ulcerative colitis, E-selectin-expressing vessels contained neutrophils attached to endothelial cells, presumably in the process of extravasation, which eventually could cause epithelial damage. These results overall indicate that U12-12 is effective for E-selectin immunohistochemistry in archived FFPE samples representing various human diseases.

Beta3Gn-T7 Is a Keratan Sulfate ß1,3 N-Acetylglucosaminyltransferase in the Adult Brain.

Keratan sulfate (KS) glycan is covalently attached to a core protein of proteoglycans. KS is abundant in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net-positive neurons in the adult brain under physiological conditions. We previously showed that the synthesis of KS positive for the R-10G antibody in the adult brain is mediated by GlcNAc-6-sulfotransferase 3 (GlcNAc6ST3; encoded by Chst5). Deficiency in both GlcNAc6ST3 and GlcNAc6ST1, encoded by Chst2, completely abolished KS. Protein-tyrosine phosphatase receptor type z1 (Ptprz1)/phosphacan was identified as a KS scaffold. KS requires the extension of GlcNAc by ß1,3 N-acetylglucosaminyltransferase (Beta3Gn-T). Members of the Beta3Gn-T family involved in the synthesis of adult brain KS have not been identified. In this study, we show by a method of gene targeting that Beta3Gn-T7, encoded by B3gnt7, is a major Beta3Gn-T for the synthesis of KS in neuropils and the perineuronal region in the adult brain. Intriguingly, the B3gnt7 gene is selectively expressed in oligodendrocyte precursor cells (OPCs) and oligodendrocytes similar to that of GlcNAc6ST3. These results indicate that Beta3Gn-T7 in oligodendrocyte lineage cells may play a role in the formation of neuropils and perineuronal nets in the adult brain through the synthesis of R-10G-positive KS-modified proteoglycan.

The Conspicuousness of High Endothelial Venules in Angioimmunoblastic T-cell Lymphoma Is Due to Increased Cross-sectional Area, Not Increased Distribution Density.

Angioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma of follicular helper T-cell origin. Histologically, neoplastic T-cells proliferate to form clusters adjacent to or between arborizing high endothelial venules (HEVs). HEVs in normal lymph nodes express sulfated glycans called peripheral lymph node addressin (PNAd); however, it remains unclear whether PNAd is also expressed on HEVs in AITL. Furthermore, although it is widely accepted that HEVs are conspicuous in AITL due to their proliferation, quantitative histological support for this concept is lacking. To investigate these issues, we employed monoclonal antibodies recognizing PNAd, namely, MECA-79, HECA-452, and 297-11A, and performed quantitative immunohistochemical analysis of HEVs in 36 AITL-affected and 67 normal lymph nodes. Staining with all three antibodies confirmed that AITL HEVs express PNAd. Moreover, AITL HEVs were bound calcium-dependently by L-selectin-IgM fusion proteins, indicating that they function in the recruitment of L-selectin-expressing lymphocytes. Unexpectedly, HEV distribution density was not increased but rather decreased in AITL compared with normal lymph nodes, but HEV cross-sectional area in AITL was significantly greater than that seen in normal lymph nodes. Overall, these results indicate that the prominence of AITL HEVs is likely due to increased cross-sectional area rather than increased distribution density.

Apical Membrane Expression of Distinct Sulfated Glycans Is a Characteristic Feature of Ductules and Their Reactive and Neoplastic Counterparts.

Intrahepatic bile ducts transport bile between bile canaliculi and the extrahepatic bile duct. The luminal surface of this tract is lined by a layer of biliary epithelial cells, or cholangiocytes, which secrete mucins consisting of scaffold proteins and O-glycosidically linked carbohydrate side chains. Although mucin core proteins have been extensively investigated, the structure and function of carbohydrate side chains have not. Here, we demonstrate that distinct sulfated glycans positive for MECA-79, R-10G, and 297-11A, but not 5D4, monoclonal antibodies are expressed in the cytoplasm of cells of large-sized ducts and in the apical membrane of cells in ductules, and that R-10G immunolabeling is partially eliminated by endo-ß-galactosidase digestion, supporting the presence of N-acetylglucosamine-6-O-sulfated N-acetyllactosamine structures. We observed comparable apical membrane-predominant staining in ductular reactions seen during regeneration that occurs in various liver diseases and in cholangiolocarcinoma, a subtype of small duct-type intrahepatic cholangiocarcinoma (iCCA). Apical membrane expression of distinct sulfated glycans in large duct-type iCCA was negligible. Intriguingly, under pathological conditions, endo-ß-galactosidase digestion almost completely eliminated R-10G immunoreactivity. These findings suggest that apical membrane expression of distinct sulfated glycans is a characteristic feature of ductules and their reactive and neoplastic counterparts.

Development of an orally-administrable tumor vasculature-targeting therapeutic using annexin A1-binding D-peptides.

We previously reported that IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminus, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the ANXA1 N-terminus as target. We then identified peptide sequences, synthesized them as D-amino acids, and designated the resulting peptide dTIT7, which we showed bound to the ANXA1 N-terminus. Whole body imaging of mouse brain tumor models injected with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered dTIT7/geldanamycin (GA) conjugates suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that ANXA1-binding D-peptide can be developed as an orally-administrable tumor vasculature-targeted therapeutic.

Simultaneous targeting of primary tumor, draining lymph node, and distant metastases through high endothelial venule-targeted delivery.

Cancer patients with malignant involvement of tumor-draining lymph nodes (TDLNs) and distant metastases have the poorest prognosis. A drug delivery platform that targets the primary tumor, TDLNs, and metastatic niches simultaneously, remains to be developed. Here, we generated a novel monoclonal antibody (MHA112) against peripheral node addressin (PNAd), a family of glycoproteins expressed on high endothelial venules (HEVs), which are present constitutively in the lymph nodes (LNs) and formed ectopically in the tumor stroma. MHA112 was endocytosed by PNAd-expressing cells, where it passed through the lysosomes. MHA112 conjugated antineoplastic drug Paclitaxel (Taxol) (MHA112-Taxol) delivered Taxol effectively to the HEV-containing tumors, TDLNs, and metastatic lesions. MHA112-Taxol treatment significantly reduced primary tumor size as well as metastatic lesions in a number of mouse and human tumor xenografts tested. These data, for the first time, indicate that human metastatic lesions contain HEVs and provide a platform that permits simultaneous targeted delivery of antineoplastic drugs to the three key sites of primary tumor, TDLNs, and metastases.

A matricellular protein fibulin-4 is essential for the activation of lysyl oxidase.

Fibulin-4 is a matricellular protein required for extracellular matrix (ECM) assembly. Mice deficient in fibulin-4 (Fbln4-/- ) have disrupted collagen and elastin fibers and die shortly after birth from aortic and diaphragmatic rupture. The function of fibulin-4 in ECM assembly, however, remains elusive. Here, we show that fibulin-4 is required for the activity of lysyl oxidase (LOX), a copper-containing enzyme that catalyzes the covalent cross-linking of elastin and collagen. LOX produced by Fbln4-/- cells had lower activity than LOX produced by wild-type cells due to the absence of lysine tyrosyl quinone (LTQ), a unique cofactor required for LOX activity. Our studies showed that fibulin-4 is required for copper ion transfer from the copper transporter ATP7A to LOX in the trans-Golgi network (TGN), which is a necessary step for LTQ formation. These results uncover a pivotal role for fibulin-4 in the activation of LOX and, hence, in ECM assembly.

Overcoming the blood-brain barrier by Annexin A1-binding peptide to target brain tumours.

BACKGROUND: Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. METHODS: (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 µmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 µmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. RESULTS: (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. CONCLUSIONS: IF7C(RR)-SN38 crosses the blood-brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.

Preferential expression of sialyl 6'-sulfo N-acetyllactosamine-capped O-glycans on high endothelial venules in human peripheral lymph nodes.

Lymphocyte "homing", the physiologic trafficking of lymphocytes from the circulation to secondary lymphoid organs, is regulated by sequential adhesive interactions between lymphocytes and endothelial cells that constitute high endothelial venules (HEVs). Initial lymphocyte "rolling" is mediated by relatively weak, transient adhesive interactions between L-selectin expressed on lymphocytes and sulfated mucin-type O-glycans expressed on HEVs. Keratan sulfate galactose (Gal)-6-O-sulfotransferase (KSGal6ST) catalyzes 6-O-sulfation of Gal in keratan sulfate glycosaminoglycan chains but also transfers sulfate to Gal in much shorter glycan chains, such as sialylated N-acetyllactosamine (LacNAc)-capped O-glycans. In mice, KSGal6ST is reportedly expressed in HEVs and functions in synthesizing 6-sulfo Gal-containing O-glycans on HEVs. However, in humans, the presence of 6-sulfo Gal-containing O-glycans on HEVs is not reported. Employing the newly developed monoclonal antibody 297-11A, which recognizes non-sialylated terminal 6'-sulfo LacNAc, we demonstrate that sialyl 6'-sulfo (and/or 6,6'-disulfo) LacNAc-capped O-glycans are preferentially displayed on HEVs in human peripheral lymph nodes (PLNs) and to a lesser extent in mesenteric LNs (MLNs) but not in Peyer's patches (PPs). We also found that the scaffold protein mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed on HEVs in PPs and MLNs but not PLNs, was modified by 297-11A-positive sulfated glycans less efficiently than was CD34. Moreover, 297-11A-positive sulfated glycans were also displayed on HEV-like vessels induced in tumor-infiltrating lymphocyte (TIL) aggregates formed in various cancers. These findings collectively indicate that 297-11A-positive sulfated glycans potentially play a role in physiologic lymphocyte homing as well as in lymphocyte recruitment under pathologic conditions.

GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain.

Keratan sulfate (KS) is a carbohydrate side chain covalently attached to extracellular proteoglycans. KS is composed of disaccharide units of 6-sulfated N-acetylglucosamine (GlcNAc) and galactose. We have previously shown that GlcNAc-6-O-sulfotransferase (GlcNAc6ST) 1 encoded by Chst2 is an enzyme necessary for the synthesis of GlcNAc-6-sulfated KS chains that are required for neuronal plasticity in the visual cortex of the mouse brain during the critical period, but not in adulthood. Here, we show that GlcNAc-6-sulfated KS recognized by the R-10G anti-KS antibody, of which the minimum epitope structure is Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S), distributes diffusely in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net (PNN)-positive neurons in the adult visual cortex. Surprisingly, GlcNAc6ST3, which was discovered as an intestinal GlcNAc6ST encoded by Chst5, is a major brain KS sulfotransferase expressed in oligodendrocytes in adulthood. Moreover, we identified an isoform of the protein-tyrosine phosphatase PTPRZ as a R-10G-reactive KS proteoglycan. These results indicate that GlcNAc6ST3 may play a role in synthesis of a component of PNN in the adult brain, and that the KS-modified isoform of PTPRZ encoded by Ptprz1 could be an extracellular molecule associated with PNNs.

Keratan Sulfate Phenotype in the ß-1,3-N-Acetylglucosaminyltransferase-7-Null Mouse Cornea.

Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine-transferring enzyme, ß-1,3-N-acetylglucosaminyltransferase-7 (ß3GnT7). A mouse model deficient in ß3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the ß3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via ß3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without ß3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.

Latent TGF-ß binding protein 2 and 4 have essential overlapping functions in microfibril development.

Microfibrils are exracellular matrix components necessary for elastic fiber assembly and for suspending lenses. We previously reported that latent TGF-ß binding protein 2 (LTBP-2), a microfibril-associated protein, is required for forming stable microfibril bundles in ciliary zonules. However, it was not understood why Ltbp2 null mice only showed an eye-specific phenotype, whereas LTBP-2 is abundantly expressed in other tissues containing microfibrils in wild type mice. Here, we show that LTBP-4, another microfibril-associated protein, compensates for the loss of LTBP-2 in microfibril formation. Ltbp2/4S double knockout (DKO) mice showed increased lethality due to emphysema, which was much more severe than that found in Ltbp4S null mice. Elastic fibers in the lungs of Ltbp2/4S DKO mice were severely disorganized and fragmented. Cultured mouse embryonic fibroblasts (MEFs) from Ltbp2/4S DKO embryos developed reduced microfibril meshwork in serum-free conditions, whereas the microfibril formation was restored by the addition of either recombinant LTBP-2 or -4. Finally, ectopic expression of LTBP-4 in the whole body restored ciliary zonule microfibril bundles in the eyes of Ltbp2 null mice. These data suggest that LTBP-2 and -4 have critical overlapping functions in forming the robust structure of microfibrils in vitro and in vivo.

Latent TGF-ß binding protein-2 is essential for the development of ciliary zonule microfibrils.

Latent TGF-ß-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.