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

Abstract

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.

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

“…Unexpectedly, mice deficient in GlcNAc6ST2 showed negligible levels of pleural R-10G signals ( Figure 3 A,B). As seen in the previous report [ 3 ], the mesothelin-positive mesothelium was thickened in GlcNAc6ST2 KO and GlcNAc6ST3 KO mice ( Figure 3 A). R-10G signals in these KO mice were not colocalized with laminins ( Supplementary Materials Figure S2 ).…”

“…We previously showed that the 6-sulfo sLe X present in the high endothelial venule (HEV) cells of peripheral lymph nodes is complementarily synthesized by GlcNAc6ST1 and GlcNAc6ST2 [ 15 , 16 ]. Recently, we reported that GlcNAc6ST2 and GlcNAc6ST3 are complementary to the synthesis of sialyl 6-sulfo LacNAc recognized by CL40 in the mouse pleura [ 3 ]. We wished to determine which GlcNAc6ST is responsible for the synthesis of R10G-reactive glycans in the mouse pleural mesothelium.…”

“…Ks 36) were obtained from Seikagaku Corporation (Tokyo, Japan). The enzyme pretreatments were optimized previously [ 3 , 37 ].…”

“…It is important to determine which GlcNAc6STs are involved in the synthesis of which sulfated glycans in a tissue-specific and cell-specific manner. Recently, we reported that sialyl 6-sulfo N -acetyllactosamine (sialyl 6-sulfo LacNAc, Neu5Acα2-3Galβ1-4(6S)GlcNAc), a sialylated GlcNAc-6-sulfated glycan recognized by the CL40 antibody [ 2 ], is present in the mesothelin-positive mesothelium of pulmonary pleura under physiological conditions [ 3 ]. We also reported that the synthesis of CL40-positive glycans in the mouse lung mesothelium requires GlcNAc6ST2 (encoded by Chst4 ) and GlcNAc6ST3 (encoded by Chst5 ) but not GlcNAc6ST1 (encoded by Chst2 ) or GlcNAc6ST4 (encoded by Chst7 ) [ 3 ].…”

“…Recently, we reported that sialyl 6-sulfo N -acetyllactosamine (sialyl 6-sulfo LacNAc, Neu5Acα2-3Galβ1-4(6S)GlcNAc), a sialylated GlcNAc-6-sulfated glycan recognized by the CL40 antibody [ 2 ], is present in the mesothelin-positive mesothelium of pulmonary pleura under physiological conditions [ 3 ]. We also reported that the synthesis of CL40-positive glycans in the mouse lung mesothelium requires GlcNAc6ST2 (encoded by Chst4 ) and GlcNAc6ST3 (encoded by Chst5 ) but not GlcNAc6ST1 (encoded by Chst2 ) or GlcNAc6ST4 (encoded by Chst7 ) [ 3 ]. GlcNAc6ST2 and GlcNAc6ST3 are complementary to the synthesis of CL40-reactive sialylated sulfated glycans in the mouse pleural mesothelium.…”