scholarly journals Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Prakash G. Kshirsagar ◽  
Mansi Gulati ◽  
Wade M. Junker ◽  
Abhijit Aithal ◽  
Gaelle Spagnol ◽  
...  
Keyword(s):  
Structure Prediction ◽  
Biologically Active ◽  
Β Subunit ◽  
Α Subunit ◽  
Functional Studies ◽  
Domain Specific ◽  
Random Coils ◽  
Simple Strategy ◽  
Egfr Family

AbstractMUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

scholarly journals Characterization of Recombinant β Subunit of Human MUC4 Mucin (rMUC4β)

2021 ◽  
Author(s):  
Prakash G. Kshirsagar ◽  
Mansi Gulati ◽  
Wade M. Junker ◽  
Abhijit Aithal ◽  
Gaelle Spagnol ◽  
...  
Keyword(s):  
Structure Prediction ◽  
Biologically Active ◽  
Β Subunit ◽  
Α Subunit ◽  
Functional Studies ◽  
Domain Specific ◽  
Random Coils ◽  
Simple Strategy ◽  
Egfr Family

Abstract MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant rMUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 721-729 ◽  
Author(s):  
Johanna VEIJOLA ◽  
Pia ANNUNEN ◽  
Peppi KOIVUNEN ◽  
Antony P. PAGE ◽  
Taina PIHLAJANIEMI ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cloning And Expression ◽  
Β Subunit ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
C Elegans ◽  
Baculovirus Expression ◽  
Expression Studies ◽  
Α Subunits

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.

scholarly journals Characterization of Hybrid Toluate and Benzoate Dioxygenases

2003 ◽  
Vol 185 (18) ◽  
pp. 5333-5341 ◽  
Author(s):  
Yong Ge ◽  
Lindsay D. Eltis
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Acinetobacter Calcoaceticus ◽  
The Other ◽  
Β Subunit ◽  
Structural Determinants ◽  
Α Subunit ◽  
Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

Heterogeneity of human luteinizing hormone. Detection and identification of α- and β-subunits in international reference preparations

1987 ◽  
Vol 114 (4) ◽  
pp. 572-576 ◽  
Author(s):  
L. A. van Ginkel ◽  
J. G. Loeber
Keyword(s):  
Luteinizing Hormone ◽  
Biologically Active ◽  
Β Subunit ◽  
Charge Heterogeneity ◽  
Α Subunit ◽  
Isoelectric Focussing ◽  
International Reference ◽  
Detection And Identification ◽  
Α And Β Subunits ◽  
Two Peaks

Abstract. The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free α-or β-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free α- or β-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the α-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the β-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM 14) which was prepared by one of us and on which we have reported earlier. In addition, with the abovementioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37°C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, α- and β-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.

scholarly journals Human Thyrotropin (TSH) Variants Designed by Site-Directed Mutagenesis Block TSH Activity in Vitro and in Vivo

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2845-2850 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Zaki Kraiem ◽  
Fuad Fares
Keyword(s):  
Chorionic Gonadotropin ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Human Thyroid ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

Abstract TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific β-subunit and a common α-subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-β subunit as a linker [hTSHβ-carboxyl-terminal peptide (CTP)-α]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the α-subunit (hTSHβ-CTP-αdeg) and the other lacking also the oligosaccharides on the β-subunit (hTSHβdeg-CTP-αdeg). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.

Expression and characterization of biologically active ovine FSH from mammalian cell lines

1994 ◽  
Vol 12 (1) ◽  
pp. 71-83 ◽  
Author(s):  
P S Mountford ◽  
M R Brandon ◽  
T E Adams
Keyword(s):  
Sertoli Cell ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Biologically Active ◽  
Expression Vectors ◽  
Β Subunit ◽  
Α Subunit ◽  
Mammalian Cell Lines

ABSTRACT Stably transfected cell lines expressing the α subunit, β subunit and α/β heterodimer of ovine (o)FSH have been established following the transfection of Chinese hamster ovary cells with α and β subunit cDNA expression vectors. In the absence of the α subunit, FSH β subunit polypeptides were inefficiently secreted and displayed a short intracellular half-life, while free α subunits were readily secreted in the absence of the β subunit. Cotransfection of oFSH α and β subunit cDNAs led to heterodimer assembly and secretion. While alteration of the nucleotide sequence flanking the β subunit AUG initiation codon did not appreciably enhance heterodimer biosynthesis and secretion, the replacement of the 5′ untranslated and signal peptide-coding regions of the β subunit cDNA with the corresponding sequences from an oGH cDNA clone was associated with a twofold increase in oFSH heterodimer secretion. The recombinant oFSH had a higher molecular weight than pituitary-derived oFSH, and was more acidic than the native hormone when analysed using isoelectric focusing, suggesting a greater degree of sialylation of the recombinant hormone. A comparison of the activities of the recombinant and native hormones in the porcine testis radioreceptor assay and in the in vitro Sertoli cell bioassay revealed that the recombinant oFSH displayed enhanced biological activity in the Sertoli cell assay when compared with the native hormone.

Heterogeneity of human lutropin. Detection and identification of α- and β-subunits

1985 ◽  
Vol 110 (2) ◽  
pp. 182-192 ◽  
Author(s):  
L. A. van Ginkel ◽  
J. G. Loeber
Keyword(s):  
Biological Activity ◽  
Isoelectric Focusing ◽  
Biologically Active ◽  
Ph Gradient ◽  
Β Subunit ◽  
Active Components ◽  
Α Subunit ◽  
Relative Response ◽  
Detection And Identification

Abstract. A highly purified LH preparation, prepared from human pituitaries (NM14) was studied using immunochemical and in vitro biological techniques. Using isoelectric focusing 5 different biologically active components could be detected, 4 of which were located between pH = 7.0 and 8.6, one was located at pH = 4.9. The biological activity in the acidic part of the pH gradient is probably due to the formation of an artefact during storage in solution. The experiments were performed with special emphasis on the occurrence of LH subunits, for which until now no pI-values have been reported. Using specific radioimmunochemical (RIA) systems at least 7 different α-subunit components and 4 different β-subunit components could be detected. The presence of even more components is likely. The α-subunit components, with pI-values of: 4.6, 5.2, 6.0, 7.1, 8.1, 8.8 and 9.7, were located spread over the entire pH-gradient whereas all β-subunit components were located above pH = 7.5, the pI-values being 7.7, 8.4, 8.5 and 10.4. The identification of these components as α- or β-subunit was based on the relative response in the different RIA systems, the absence of biological activity and the response changes during incubation at 37°C. Refocusing of the above mentioned biologically active components individually resulted each time in a single component with a pI-value identical to its corresponding 'parent'. After incubation at 37°C of these components each time the same variety of subunit components was found with discrete pI-values, identical to those above.

scholarly journals Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulusP6 Biphenyl Dioxygenase and of Chimeras Derived from It

1999 ◽  
Vol 181 (16) ◽  
pp. 4805-4811 ◽  
Author(s):  
Hervé Chebrou ◽  
Yves Hurtubise ◽  
Diane Barriault ◽  
Michel Sylvestre
Keyword(s):  
The Other ◽  
Substrate Selectivity ◽  
Β Subunit ◽  
Comamonas Testosteroni ◽  
Biphenyl Dioxygenase ◽  
Α Subunit ◽  
E Coli ◽  
Hybrid Enzymes ◽  
Catalytic Capacity

ABSTRACT In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The α or β subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, ht-αLB400βP6, and ht-αB-356βP6. ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356 were not expressed active in recombinant Escherichia coli cells carrying P6bphA1 and bphA2, P6 bphA1 and LB400bphE, or P6 bphA1 and B-356 bphEbecause the [2Fe-2S] Rieske cluster of P6 oxygenase α subunit was not assembled correctly in these clones. On the other hand ht-αLB400βP6 and ht-αB-356βP6 were produced active inE. coli. Furthermore, active purified ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putidaKT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins inPseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an α and a β subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

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