Using Secretion to Solve a Solubility Problem: High-Yield Expression in Escherichia coli and Purification of the Bacterial Glycoamidase PNGase F

Loo, Trevor S.; Patchett, Mark L.; Lott, J.S.

doi:10.1006/prep.2001.1555

Cited by 46 publications

(33 citation statements)

References 21 publications

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“…Total cell lysates were digested with either PNGase F (synthesised at the School of Biological Sciences, University of Auckland, Loo et al (2002)), endoglycosidase H or neuraminidase (New England Biolabs, Ipswich, MA, USA). Lysates (100 mg) were digested with 2 mg of PNGase F in PBSC1% SDS for 4 h at 37 8C or with 500 U of endoglycosidase H or 50 U of neuraminidase according to the manufacturer's instructions for 16-18 h at 37 8C.…”

Section: Analysis Of N-linked Glycosylation and Sialylationmentioning

confidence: 99%

hMRAPa specifically alters hMC4R molecular mass and N-linked complex glycosylation in HEK293 cells

Kay¹,

Botha²,

Montgomery³

et al. 2013

Journal of Molecular Endocrinology

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Human melanocortin 2 receptor accessory protein 1(hMRAPa) is essential for human melanocortin 2 receptor (hMC2R)-regulated adrenal steroidogenesis. hMRAPa enhances hMC2R N-linked glycosylation and maturation, promotes hMC2R cell surface expression and enables ACTH to bind and activate the MC2R. However, hMRAPa is predicted to have functions beyond its critical role in hMC2R activity. It is more widely expressed than the hMC2R and it has been shown to co-immunoprecipitate with all other hMCR subtypes and other G-protein-coupled receptors, when these are co-expressed with each receptor in heterologous cells. The physiological relevance of hMRAPa interactions with these receptors is unknown. We hypothesised that hMRAPa could influence post-translational processing and maturation of these receptors, similar to its actions on the hMC2R. Here we used co-immunoprecipitation and western blotting techniques to characterise effects of hMRAPa-FLAG co-expression on the maturation of each HA-tagged hMCR subtype and the HA-tagged human calcitonin receptor-like receptor (hCL), co-expressed in HEK293 cells. While hMRAPa-FLAG interacted with all five HA-hMCR subtypes and the HA-hCL, it only altered HA-hMC4R molecular mass. This altered HA-hMC4R molecular mass was due to a change in endoglycosidase H-resistant complex N-linked glycosylation, which we observed for HA-hMC4R in both intracellular and cell surface fractions. This effect was specific to the HA-hMC4R as hMRAPa did not alter the molecular mass of any of the other receptors that we examined. In conclusion, the specific effects of hMRAPa on hMC4R molecular mass and complex N-linked glycosylation provide evidence in support of a role for MRAPa in hMC4R functions.

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Section: Analysis Of N-linked Glycosylation and Sialylationmentioning

confidence: 99%

hMRAPa specifically alters hMC4R molecular mass and N-linked complex glycosylation in HEK293 cells

Kay¹,

Botha²,

Montgomery³

et al. 2013

Journal of Molecular Endocrinology

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“…Deglycosylation of Ani-PME1 and Ani-PME2-Removal of N-linked high mannose glycans from Ani-PME was performed by incubation with maltose-binding protein-tagged EndoH f (New England Biolabs) or His-tagged PNGaseF (81). For EndoH f reactions, the sample was deglycosylated in 1ϫ G5 Reaction Buffer with 40,000 units of EndoH f per ml of sample and incubated for 20 h at 37°C.…”

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confidence: 99%

Structure and Properties of a Non-processive, Salt-requiring, and Acidophilic Pectin Methylesterase from Aspergillus niger Provide Insights into the Key Determinants of Processivity Control

Kent

Loo

Melton

et al. 2016

Journal of Biological Chemistry

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Many pectin methylesterases (PMEs) are expressed in plants to modify plant cell-wall pectins for various physiological roles. These pectins are also attacked by PMEs from phytopathogens and phytophagous insects. The de-methylesterification by PMEs of the O6-methyl ester groups of the homogalacturonan component of pectin, exposing galacturonic acids, can occur processively or non-processively, respectively, describing sequential versus single de-methylesterification events occurring before enzyme-substrate dissociation. The high resolution x-ray structures of a PME from Aspergillus niger in deglycosylated and Asn-linked N-acetylglucosamine-stub forms reveal a 10 2 ⁄ 3-turn parallel ␤-helix (similar to but with less extensive loops than bacterial, plant, and insect PMEs). Capillary electrophoresis shows that this PME is non-processive, halophilic, and acidophilic. Molecular dynamics simulations and electrostatic potential calculations reveal very different behavior and properties compared with processive PMEs. Specifically, uncorrelated rotations are observed about the glycosidic bonds of a partially de-methyl-esterified decasaccharide model substrate, in sharp contrast to the correlated rotations of processive PMEs, and the substrate-binding groove is negatively not positively charged.The heterogeneous polysaccharide pectin (1-4) is a key component of the plant cell wall (5). A host of enzymes, including pectin methylesterase (PME) 3 as well as endo-and exopolygalacturonases, modifies pectin fine-structure locally in space and time for purposes of plant-cell differentiation, cell adhesion/separation, growth, and development from roots to meristem, morphogenesis, seed and fruit development, and defense against pathogens (6 -16). PMEs hydrolyze the O6-methyl ester groups of the homogalacturonan (HG) chains that form the backbone of pectin (Fig. 1). As well as being ubiquitous in plants, and frequently manifesting in a staggering number of isoforms (more than 66 for Arabidopsis thaliana) (8, 10), PMEs are found in phytopathogenic bacteria and fungi (17-26) and in Archaea (especially Halobacteriaceae (27)), and they have also been observed in the genomes of several phytophagous insects (28 -30) and in human gut microflora (31). Recently, plant pollen PMEs have been identified as a key human allergen (32, 33). Bacterial and plant PMEs that have been functionally characterized are generally observed to be processive (34 -55), i.e. the enzyme binds to the HG substrate and then successively hydrolyzes a block of methyl ester groups before dissociating. In all cases characterized to date, the direction of processivity is from the non-reducing end of the HG chain toward the reducing end, as shown in Fig. 1. The degree of processivity (also called the action pattern) has been reported to be dependent on ionic strength and pH (56), and various classifications of PMEs exist (48,49,(57)(58)(59)(60). In general, processive PMEs have their isoelectric points, pI, and pH for optimum activity at near-neutral to basic pH, although ...

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“…Ribonuclease A (RA), Ribonuclease B (RB), Ovalbumin (OVA), chondroitin sulfate (CS), and Biotin-LC-NHS were purchased from Sigma. PNGase F was expressed and purified according to a previously published procedure [39] . Table S1).…”

Section: Methodsmentioning

confidence: 99%

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

et al. 2016

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CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAINtruncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from ...

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Using Secretion to Solve a Solubility Problem: High-Yield Expression in Escherichia coli and Purification of the Bacterial Glycoamidase PNGase F

Cited by 46 publications

References 21 publications

hMRAPa specifically alters hMC4R molecular mass and N-linked complex glycosylation in HEK293 cells

hMRAPa specifically alters hMC4R molecular mass and N-linked complex glycosylation in HEK293 cells

Structure and Properties of a Non-processive, Salt-requiring, and Acidophilic Pectin Methylesterase from Aspergillus niger Provide Insights into the Key Determinants of Processivity Control

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

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