Endo‐β‐Galactosidases and Keratanase

Fukuda, Michiko N.

doi:10.1002/0471142727.mb1717bs32

Cited by 6 publications

(8 citation statements)

References 28 publications

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“…These oligosaccharides were radiolabeled by NaB[ 3 H] 4 reduction. Exoglycosidases and endoglycosidases used for structural analysis were: β‐galactosidase, β‐ N ‐acetylhexosaminidase and α‐mannosidase from jack bean (Seikagaku, Japan), and endo‐β‐galactosidase from Escherichia freundii [18].…”

Section: Methodsmentioning

confidence: 99%

Overexpression of the Golgi‐localized enzyme α‐mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl‐N‐acetylchitobiose to tetramannosyl‐N‐acetylchitobiose in the N‐glycan‐processing pathway

Oh-eda

Nakagawa

Akama

et al. 2001

European Journal of Biochemistry

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Keywords: a-mannosidase II; a-mannosidase IIx; Golgi; HPLC; N-glycan.The major biosynthetic pathway of N-glycans in higher organisms has been established [1]. In the endoplasmic reticulum (ER), a lipid-linked glucosylated high-mannose oligosaccharide is transferred from the dolichol donor to a protein acceptor. High-mannose oligosaccharides are then processed by a-glucosidases and a-1,2 mannosidases (MI) to produce Man 5 GlcNAc 2 (M 5 Gn 2 ). A key conversion of this oligosaccharide to complex-type oligosaccharides occurs in the medial Golgi, where N-acetylglucosaminyltransferase-I (GnT-I) adds N-acetylglucosamine to the Mana133Manb13 terminal of M 5 Gn 2 to form Gn 1 M 5 Gn 2 [2]. The Golgi a-mannosidase II (MII) then removes two mannosyl residues from Gn 1 M 5 Gn 2 to form Gn 1 M 3 Gn 2 [3], which are further modified to complex oligosaccharides by N-acetylglucosaminyltransferase-II (GnT-II), galactosyltransferase and sialyltransferases.The presence of an MII-related enzyme was suggested by studies on a human genetic disease, congenital dyserythropoietic anemia type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test). Although the primary gene defect of HEMPAS is heterogeneous [4±8], the biochemical phenotype is characterized as a failure in N-glycan processing. Furthermore, mutant mice in which the MII gene was inactivated by homologous recombination showed strikingly similar phenotypes to those exhibited by HEMPAS patients [9]. Although cells from MII-null mice completely lack MII activity and accumulate hybrid-type oligosaccharides, MII-null splenocytes and fibroblasts were found to synthesize complex-type oligosaccharides. This finding indicates that complex oligosaccharides can be synthesized by an MII-independent pathway, leading to the proposal of an alternative pathway and the prediction of an a-mannosidase, designated a-mannosidase III (MIII). MIII hydrolyzes two a-mannosyl residues in M 5 Gn 2 and produces M 3 Gn 2 , which is further converted to Gn 1 M 3 Gn 2 by GnT-I. Bonay and Hughes [10] found a novel a-mannosidase in rat liver. This enzyme, in a cobalt iondependent manner, hydrolyzed a132, a133, and a136 mannosyl linkages of N-glycans. As the MIII activity was not observed in the tissues of the MII-null mouse until cobalt was added, MIII is most certainly the same activity Eur.

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Section: Methodsmentioning

confidence: 99%

Overexpression of the Golgi‐localized enzyme α‐mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl‐N‐acetylchitobiose to tetramannosyl‐N‐acetylchitobiose in the N‐glycan‐processing pathway

Oh-eda

Nakagawa

Akama

et al. 2001

European Journal of Biochemistry

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“…This DPC-generated KSPG is, in fact, larger than that produced by human corneal stromal stem cells in vitro [44] and in the same size range reported for KSPG synthesized in intact human corneal tissue [45]. The immunoblot in Figure 1 was developed with a monoclonal antibody specific for sulfated epitopes on the KS chains [46], and the keratanase enzyme that removed immunoreactivity from KSPG requires sulfation of the KS chain for glycolytic cleavage of the polysaccharide chain [47]. Together, these data provide inescapable evidence that the DPCs are producing sulfated KS chains of a molecular size consistent with that in normal human cornea.…”

Section: Discussionmentioning

confidence: 86%

Dental Pulp Stem Cells: A New Cellular Resource for Corneal Stromal Regeneration

Syed-Picard

Lathrop

et al. 2015

Stem Cells Translational Medicine

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Corneal blindness afflicts millions of individuals worldwide and is currently treated by grafting with cadaveric tissues; however, there are worldwide donor tissue shortages, and many allogeneic grafts are eventually rejected. Autologous stem cells present a prospect for personalized regenerative medicine and an alternative to cadaveric tissue grafts. Dental pulp contains a population of adult stem cells and, similar to corneal stroma, develops embryonically from the cranial neural crest. We report that adult dental pulp cells (DPCs) isolated from third molars have the capability to differentiate into keratocytes, cells of the corneal stoma. After inducing differentiation in vitro, DPCs expressed molecules characteristic of keratocytes, keratocan, and keratan sulfate proteoglycans at both the gene and the protein levels. DPCs cultured on aligned nanofiber substrates generated tissue-engineered, corneal stromal-like constructs, recapitulating the tightly packed, aligned, parallel fibrillar collagen of native stromal tissue. After injection in vivo into mouse corneal stroma, human DPCs produced corneal stromal extracellular matrix containing human type I collagen and keratocan and did not affect corneal transparency or induce immunological rejection. These findings demonstrate a potential for the clinical application of DPCs in cellular or tissue engineering therapies for corneal stromal blindness.

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“…We then investigated whether the enzymatic removal of GlcNAc-6-sulfated or non-sulfated poly-LacNAc [ 42 , 43 ] could abolish R-10G immunoreactivity in the pleura. The pretreatment of lung sections with endo-β-galactosidase could abolish R-10G immunoreactivity ( Figure 2 A,B).…”

Section: Resultsmentioning

confidence: 99%

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

Takeda-Uchimura,

Ikezaki,

Akama

et al. 2024

Molecules

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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.

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Endo‐β‐Galactosidases and Keratanase

Abstract: This overview covers the endo-beta-galactosidases; enzyme is capable of hydrolyzing a wide range of glycoconjugates. Endo-beta-galactosidases from numerous sources are discussed in terms of their substrate specificities and substrates, as well as their practical research applications.

Cited by 6 publications

References 28 publications

Overexpression of the Golgi‐localized enzyme α‐mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl‐N‐acetylchitobiose to tetramannosyl‐N‐acetylchitobiose in the N‐glycan‐processing pathway

Overexpression of the Golgi‐localized enzyme α‐mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl‐N‐acetylchitobiose to tetramannosyl‐N‐acetylchitobiose in the N‐glycan‐processing pathway

Dental Pulp Stem Cells: A New Cellular Resource for Corneal Stromal Regeneration

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

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