The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

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Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

Biochemical Journal ◽

10.1042/bj3020095 ◽

1994 ◽

Vol 302 (1) ◽

pp. 95-101 ◽

Cited By ~ 11

Author(s):

A Schejter ◽

T I Koshy ◽

T L Luntz ◽

R Sanishvili ◽

I Vig ◽

...

Keyword(s):

Cytochrome C ◽

Site Directed Mutagenesis ◽

Wild Type ◽

General Increase ◽

Reduction Potentials ◽

Cytochromes C ◽

In The Wild ◽

Order Of Magnitude ◽

The Stability ◽

Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

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Effects of N224 glycosylation in Saccharomycopsis fibuligera R64 α-amylase on enzyme activity and stability

Current Enzyme Inhibition ◽

10.2174/1573408017666210809111830 ◽

2021 ◽

Vol 17 ◽

Author(s):

Yovin Sugijo ◽

Tina Dewi Rosahdi ◽

Fernita Puspasari ◽

Wangsa Tirta Ismaya ◽

Khomaini Hasan ◽

...

Keyword(s):

Thermal Stability ◽

Enzyme Activity ◽

Evolutionary Relationship ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Saccharomycopsis Fibuligera ◽

Glycosylation Sites ◽

Starch Substrate ◽

Relationship Of

Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains the two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. Objective: To test whether N224 plays a key role in enzyme activity and stability. Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activity of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

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Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.591-598.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 591-598 ◽

Cited By ~ 34

Author(s):

Ulrike Pag ◽

Christoph Heidrich ◽

Gabriele Bierbaum ◽

Hans-Georg Sahl

Keyword(s):

Gene Cluster ◽

Inducible Promoter ◽

Biosynthetic Gene Cluster ◽

Site Directed Mutagenesis ◽

Start Codon ◽

Wild Type ◽

Tightly Coupled ◽

In The Wild ◽

The Stability ◽

Self Protection

ABSTRACT The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Within its biosynthetic gene cluster, the immunity gene pepI, providing producer self-protection, is localized upstream of the structural gene pepA. Pep5 production and the immunity phenotype have been found to be tightly coupled (M. Reis, M. Eschbach-Bludau, M. I. Iglesias-Wind, T. Kupke, and H.-G. Sahl, Appl. Environ. Microbiol. 60:2876–2883, 1994). To study this phenomenon, we analyzed pepA and pepItranscription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing different levels of immunity. Complementation of apepA-expressing strain with pepI intrans did not result in phenotypic immunity or production of PepI. On the other hand, neither pepA nor its product was found to be involved in immunity, since suppression of the translation of the pepA mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression of pepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability ofpepI transcripts, which in the wild-type strain, is achieved by an inverted repeat with a free energy of −56.9 kJ/mol, localized downstream of pepA. We performed site-directed mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immunity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently was less efficient in providing self-protection than the wild-type PepI.

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Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin

Acta Endocrinologica ◽

10.1530/eje.0.1320611 ◽

1995 ◽

Vol 132 (5) ◽

pp. 611-617 ◽

Cited By ~ 10

Author(s):

Marcel T den Hartog ◽

Carin C Sijmons ◽

Onno Bakker ◽

Carrie Ris-Stalpers ◽

Jan JM de Vijlder

Keyword(s):

Amino Acid ◽

Medical Center ◽

Academic Medical Center ◽

Negative Control ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Terminal Part ◽

Tyrosine Residues ◽

In The Wild

Den Hartog MT, Sijmons CC, Bakker 0, Ris-Stalpers C, de Vijlder JJM. Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin. Eur J Endocrinol 1995;132:611–17. ISSN 0804–4643 Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment. This indicates that a preferential involvement of tyrosines 5 and 130 in thyroid hormonogenesis may exist. JJM de Vijlder, Academic Medical Center, University of Amsterdam, Children's Hospital "Emma Kinderziekenhuis/Het Kinder AMC", PO Box 22700, 1100 DE Amsterdam, The Netherlands

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Lecithin:cholesterol acyltransferase: role of N-linked glycosylation in enzyme function

Biochemical Journal ◽

10.1042/bj2940879 ◽

1993 ◽

Vol 294 (3) ◽

pp. 879-884 ◽

Cited By ~ 29

Author(s):

K O ◽

J S Hill ◽

X Wang ◽

R McLeod ◽

P H Pritchard

Keyword(s):

Enzyme Activity ◽

Specific Activity ◽

Consensus Sequence ◽

Fold Increase ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Lecithin:Cholesterol Acyltransferase ◽

Mutant Proteins ◽

Glycosylation Sites

Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is a glycoprotein which is responsible for the formation of cholesteryl ester in plasma. The carbohydrate content has been estimated to be approx. 25% of the total LCAT mass, and four potential N-linked glycosylation sites have been predicted at residues 20, 84, 272 and 384 of the LCAT protein sequence. In the present study, we have examined which of these sites are utilized and how the N-glycosylation affects the secretion and function of the enzyme. Site-directed mutagenesis was performed to eliminate the glycosylation consensus sequence at each of the four potential sites, and the mutant proteins were expressed in COS cells. The amount of each mutant LCAT secreted was similar to that of the wild-type enzyme but the molecular mass was decreased by 3-4 kDa. The specific activity of each mutant LCAT was significantly different from the wild-type; however, the magnitude and direction of the change depended on the glycosylation site mutagenized. Loss of carbohydrate at position 20, 84 or 272 resulted in a decrease in the specific activity of the mutant enzymes by 18%, 82%, and 62% respectively. In contrast, the mutant protein without glycosylation at position 384 displayed a 2-fold increase in enzyme activity. In addition, a quadruple mutant was constructed such that all four potential glycosylation sites were eliminated. The amount of the unglycosylated LCAT secreted into the culture medium was less than 10% of the wild-type level and the specific activity of this enzyme was decreased to 5% of that of the wild type. The results demonstrate that all four potential N-glycosylation sites in LCAT are used and the presence of carbohydrate at each site has diverse effects on the enzyme activity.

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The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

Biochemical Journal ◽

10.1042/bj3180583 ◽

1996 ◽

Vol 318 (2) ◽

pp. 583-590 ◽

Cited By ~ 50

Author(s):

Fumio SAKANE ◽

Masahiro KAI ◽

Ikuo WADA ◽

Shin-ichi IMAI ◽

Hideo KANOH

Keyword(s):

Enzyme Activity ◽

Binding Sites ◽

Enzyme Activities ◽

Catalytic Domain ◽

Zinc Fingers ◽

Diacylglycerol Kinase ◽

Site Directed Mutagenesis ◽

Atp Binding ◽

Wild Type

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

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InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922284117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19245-19253 ◽

Cited By ~ 1

Author(s):

Soumyadip Sahu ◽

Zhenzhen Wang ◽

Xinfu Jiao ◽

Chunfang Gu ◽

Nikolaus Jork ◽

...

Keyword(s):

Stress Responses ◽

Messenger Rna ◽

Control Process ◽

Stem Cell Differentiation ◽

Wild Type ◽

Inositol Pyrophosphate ◽

Body Dynamics ◽

Processing Body ◽

The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

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Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽

10.1182/blood-2012-07-441824 ◽

2012 ◽

Vol 120 (16) ◽

pp. 3336-3344 ◽

Cited By ~ 36

Author(s):

Anu Laitala ◽

Ellinoora Aro ◽

Gail Walkinshaw ◽

Joni M. Mäki ◽

Maarit Rossi ◽

...

Keyword(s):

Cultured Cells ◽

Mrna Level ◽

Hypoxia Inducible Factor ◽

Mrna Levels ◽

Wild Type ◽

Α Subunit ◽

Hepcidin Expression ◽

In The Wild ◽

Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

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Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3020355 ◽

1994 ◽

Vol 302 (2) ◽

pp. 355-361 ◽

Cited By ~ 31

Author(s):

K Inukai ◽

T Asano ◽

H Katagiri ◽

M Anai ◽

M Funaki ◽

...

Keyword(s):

Glucose Transporter ◽

Cytochalasin B ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Site Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

In The Wild ◽

Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

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Chloroethyl urea derivatives block tumour growth and thioredoxin-1 nuclear translocation

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y10-084 ◽

2010 ◽

Vol 88 (11) ◽

pp. 1102-1114 ◽

Cited By ~ 5

Author(s):

Alexandre Patenaude ◽

Jessica S. Fortin ◽

Réna Deschenes ◽

Marie-France Côté ◽

Jacques Lacroix ◽

...

Keyword(s):

Anticancer Activity ◽

Nuclear Translocation ◽

Human Cancer ◽

Alkylating Agents ◽

Covalent Binding ◽

Intracellular Localization ◽

Site Directed Mutagenesis ◽

Loss Of Function ◽

Thioredoxin 1

Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that 14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.

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