Sequencing, expression, and enzymatic characterization of  -hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells

Andersson, Sofia V.

doi:10.1093/glycob/cwi006

Cited by 10 publications

(3 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, lysosomal proteins may be secreted into the medium, present in the secretory pathway compartment, or active in the lysosomes depending on cell type, culture conditions, and expression levels (55). As a result, it was not surprising to observe Sf hex, a homolog of mammalian hexosaminidase, in both the intracellular and secreted fractions in this study.…”

Section: Discussionmentioning

confidence: 69%

“…Interestingly, HexB, normally a lysosomal protein, is used as a marker for regulated secretion in some mammalian cell lines, and its extracellular release can parallel total protein secretion in stimulated cell lines (55). Thus, lysosomal proteins may be secreted into the medium, present in the secretory pathway compartment, or active in the lysosomes depending on cell type, culture conditions, and expression levels (55).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a ␤-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a ␤-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the ␤(1,2)-linked N-acetylglucosamine from the Man␣(1,3) branch compared with the Man␣(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal ␤-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing ␤-Nacetylhexosaminidase acting on N-glycans from Sf9 cells.␤-N-Acetylhexosaminidase (hexosaminidase, EC 3.2.1.52) catalyzes the hydrolysis of nonreducing terminal N-acetyl-Dhexosamine residues in N-acetyl-␤-D-hexosaminides. Hexosaminidases belong to the glycosyl hydrolase (GH) 2 3, GH20, or GH84 family (1-4). Of these, family 20 hexosaminidases include mammalian lysosomal hexosaminidases, fungal exochitinases, bacterial chitobiases, and insect chitinolytic hexosaminidases.The hexosaminidase activity of insects and insect cells is of particular interest because of the role that the enzyme may play in altering the structures of N-glycans generated by these cells. The N-glycan synthesis pathway in insects differs from that in mammals in that insects and insect cells produce appreciable amounts of paucimannosidic glycans (reviewed in Ref. 5). The intracellular N-glycan processing pathway in the endoplasmic reticulum of insects has been observed to include the addition of a Glc 3 Man 9 GlcNAc 2 group to the acceptor Asn residue, followed by the subsequent trimming of the initial oligosaccharide to generate Man 5 GlcNAc 2 . Insect cells also contain significant levels of N-acetylglucosaminyltransferase I, which adds a GlcNAc residue to the Man␣(1,3) branch, followed by the removal of two Man residues to produce the GlcNAc␤(1,2)Man␣(1,3)(Man(1,6))-Man␤(1,4)GlcNAc␤(1,4)GlcNAc structure. Unlike ma...

show abstract

Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Supernatants were then centrifuged at 13,000 x g for 5 min and the aspirated supernatants were stored at -20°C. Secretion was measured using a marker for secretion, β-hexosaminidase [6]; the methodology is described elsewhere [9]. In brief, a substrate for β-hexosaminidase was added to the supernatant, yielding a fluorescent product which was measured in a Flurolog 3-22 or Fluoromax 3 fluorometer (Horiba Jobin Yvon, Edison, NJ) with excitation at 365 nm and emission at 460 nm.…”

Section: In Vitro Secretion Assaymentioning

confidence: 99%

p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland

Carlsson

Gierow

2012

Cellular and Molecular Biology Letters

View full text Add to dashboard Cite

Abstract:The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and β, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

show abstract

Integrin adhesion in regulation of lacrimal gland acinar cell secretion

Andersson¹,

Gierow²

2006

Experimental Eye Research

View full text Add to dashboard Cite

Sequencing, expression, and enzymatic characterization of -hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells

Cited by 10 publications

References 39 publications

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland

Integrin adhesion in regulation of lacrimal gland acinar cell secretion

Contact Info

Product

Resources

About