scholarly journals A Novel Follitropin Analog Inhibits Follitropin Activity In Vitro

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 325
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Fuad Fares
Keyword(s):  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Fsh Receptor ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Reproductive Process ◽  
Α Subunit ◽  
Human Choriogonadotropin ◽  
Clinical Conditions

Follitropin (FSH) is a heterodimeric protein composed of an α subunit that is shared with the glycoprotein hormone family, including lutropin (LH), thyrotropin (TSH), human choriogonadotropin (hCG), and a unique β specific subunit. Both α and FSHβ subunits contain two sites of N-linked oligosaccharides, which are important for its function. FSH has a crucial function in the reproductive process in mammals. However, there are some clinical conditions, such as menopausal osteoporosis or adiposity, associated with increased FSH activity. Moreover, in some cases, carcinogenesis is evidently associated with activation of FSH receptor. Therefore, developing a follitropin antagonist might be beneficial in the treatment of these conditions. Here, we describe a novel, engineered, non-glycosylated single-chain FSH variant, prepared by site-directed mutagenesis and fusion of the coding genes of the α and β subunits. The designed variant was expressed in Chinese hamster ovary (CHO) cells and successfully secreted into the culture medium. We found that the non-glycosylated single-chain FSH analog binds with high affinity to FSH receptor and efficiently inhibits FSH activity in vitro. This variant acts at the receptor level and has the potential to serve as a follitropin antagonist for clinical applications in the future.

scholarly journals Conversion of TSH Heterodimer to a Single Polypeptide Chain Increases Bioactivity and Longevity

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 954-960 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Fuad Fares
Keyword(s):  
Binding Affinity ◽  
Half Life ◽  
Chinese Hamster ◽  
Β Subunit ◽  
Wild Type ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T4 in circulation after 2 and 4 h of treatment. However, the level of T4 after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

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.

scholarly journals Dissociation of Early Folding Events from Assembly of the Human Lutropin β-Subunit

1998 ◽  
Vol 12 (10) ◽  
pp. 1640-1649
Author(s):  
Mesut Muyan ◽  
Raymond W. Ruddon ◽  
Sheila E. Norton ◽  
Irving Boime ◽  
Elliott Bedows
Keyword(s):  
Cho Cells ◽  
Protein Disulfide Isomerase ◽  
Chinese Hamster ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Disulfide Isomerase ◽  
The Common ◽  
Protein Disulfide ◽  
Gene Expressing

Abstract The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common α-subunit and β-subunits that confer biological specificity. LHβ and CGβ share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHβ assembles with the α-subunit more slowly than does hCGβ, and only a fraction of the LHβ synthesized is secreted, whereas CGβ is secreted efficiently. To understand why the assembly and secretion of these related β-subunits differ, we studied the folding of LHβ in CHO cells transfected with either the LHβ gene alone, or in cells cotransfected with the gene expressing the common α-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pβ1, to the second major folding form, pβ2, did not differ significantly from the pβ1-to-pβ2 conversion of CGβ, suggesting that variations between the intracellular fates of the two β-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGβ, where the folding to pβ2 results in an assembly-competent product, apparently greater than 90% of the LH pβ2 recovered from LHβ-transfected CHO cells was assembly incompetent, accounting for inefficient LHβ assembly with the α-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGβ, all 12 LHβ cysteine residues formed S-S linkages as soon as pβ2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pβ2 and excess urinary α-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGβ, LHβ homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respectiveβ -subunits.

scholarly journals The Relationship between Steroidogenic Factor 1 and DAX-1 Expression and in Vitro Gonadotropin Secretion in Human Pituitary Adenomas1

2001 ◽  
Vol 86 (6) ◽  
pp. 2476-2483
Author(s):  
S. J. B. Aylwin ◽  
J. P. Welch ◽  
C. L. Davey ◽  
J. F. Geddes ◽  
D. F. Wood ◽  
...  
Keyword(s):  
Pituitary Adenomas ◽  
Messenger Rna ◽  
Gene Promoters ◽  
Orphan Nuclear Receptors ◽  
Glycoprotein Hormone ◽  
Human Pituitary ◽  
Gonadotropin Secretion ◽  
Steroidogenic Factor 1 ◽  
Α Subunit

The orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, are involved in gonadotroph differentiation, and SF-1 has been shown to activate the LH-β and glycoprotein hormone α-subunit (αGSU) gene promoters. Pituitary adenomas from 34 patients [13 somatotroph tumors, 4 prolactinomas, and 17 clinically nonfunctioning pituitary adenomas (NFPAs)] were enzymatically dispersed and cultured in vitro for 48 h. Tissue culture medium was collected and assayed for LH, FSH, and αGSU; messenger RNA was extracted from adherent cells, and expression of SF-1 and DAX-1 messenger RNA was determined by RT-PCR and verified by direct DNA sequencing. The presence of DAX-1 protein in tumor tissue was confirmed by immunocytochemistry. DAX-1 was demonstrated in all NFPAs, 7 of 13 somatotroph tumors and 0 of 4 prolactinomas. SF-1 expression occurred in 8 of 16 NFPAs, 4 of 12 somatotroph tumors, and 1 of 4 prolactinomas. LH secretion in vitro was greater in NFPAs that were SF-1 positive (P < 0.05). Neither FSH secretion nor αGSU secretion in vitro were significantly related to the expression of SF-1 or DAX-1. SF-1-positive somatotroph tumors immunostained positively for LH-β and/or FSH-β and secreted gonadotropins in vitro. SF-1 expression is associated with the in vitro secretion of LH by NFPAs. A proportion of somatotroph tumors also express SF-1 and DAX-1 and secrete gonadotropin hormones in vitro.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

158 PRODUCTION OF RECOMBINANT LH FOR USE IN TIGER (TIGRIS ALTAICA) ASSISTED REPRODUCTION:A PRELIMINARY REPORT

2008 ◽  
Vol 20 (1) ◽  
pp. 159
Author(s):  
J. T. Aaltonen ◽  
E. Bedows ◽  
K. A. Estes ◽  
V. Y. Butnev ◽  
G. Bousfield ◽  
...  
Keyword(s):  
Leydig Cell ◽  
Chinese Hamster ◽  
Granulosa Cell Tumor ◽  
Methionine Sulfoximine ◽  
Β Subunit ◽  
Wild Type ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Cell Assay ◽  
Cell Assays

Genetically, porcine LH is the closest commercially available gonadotropin analog to tiger LH (93% homologous); however, its use may lead to possible autoimmune reactions, lessening ovarian responses in stimulated tigresses over time (Crichton et al. 2005 Biol. Reprod. 68, 105–113). To overcome this problem for use in assisted reproduction, we produced recombinant tiger LH (tLH), and tested the bioactivity of several tLH constructs using heterologous (rat) and homologous (cat) Leydig cell assays. To clone tLH, mRNA was isolated from an Amur tiger pituitary by TRIzol extraction (Invitrogen, Carlsbad, CA). DNA was synthesized from the mRNA using reverse transcriptase (Stratagene, La Jolla, CA) and PCR was performed using tiger-specific primers for glycoprotein hormone α subunit or LH β subunit. The α subunit was cloned into the double-expression vector pIRES (Invitrogen). The tLH β subunit was cloned into the second site of pIRES and also into the plasmid pGS. Chinese hamster ovary (CHO) K1 and human granulosa cell tumor (COV 434) cells were transfected with plasmid DNA by calcium-phosphate precipitation: (1) pIRES containing α and pGS containing LH β, or (2) pIRES containing α and LH β. Cells were grown in selection media (250 µg mL–1 geneticine for pIRES, or 25 µm methionine sulfoximine for pGS). Media was collected and clarified at 1500g for 30 min. An immature rat Leydig cell assay protocol (Bousfield et al. 2001 Biol. Reprod. 64, 136–147) detected biological activity (testosterone production) by RIA. Of 14 tLH constructs created, 1 wild-type construct (LH WTCHO8) had LH activity 3 times greater than any other. A domestic kitten Leydig cell assay was performed in order to assess comparative sensitivities and specificities. Domestic kitten testicles, obtained from a local spay clinic, were disassociated with collagenase (225 U mg–1, Worthington Biochemical, Lakewood, NJ); however, the cells were more difficult to disperse than rat testicles, leading to low Leydig cell yields as determined histologically. Modification of the Leydig cell collagenase protocol for the cat was achieved by increasing the temperature and surface area, and agitating the minced tissue in medium on a stir plate. Samples of the 14 tLH constructs were run in parallel using rat and cat Leydig cell assays. Although rat Leydig cell testosterone concentrations (3.4 ng mL–1) were nearly 10-fold greater, the same trend for the different constructs was found in the cat Leydig cells with the same wild-type construct (LH WTCHO8, 0.37 ng mL–1) having greater LH activity than any other. The lower testosterone concentrations in the cat bioassay may be explained by insufficient Leydig cell numbers, age (sensitivity), damaging effect(s) of collagenase, or felid specificity. Still, these results lend validity to the use of the heterologous rat Leydig cell bioassay for recombinant tiger LH.

scholarly journals A Naturally Occurring Genetic Variant in the Human Chorionic Gonadotropin-β Gene 5 Is Assembly Inefficient*

Endocrinology ◽  
1999 ◽  
Vol 140 (8) ◽  
pp. 3496-3506 ◽  
Author(s):  
Amanda K. Miller-Lindholm ◽  
Elliott Bedows ◽  
Cynthia F. Bartels ◽  
Jacques Ramey ◽  
Victoria Maclin ◽  
...  
Keyword(s):  
Genetic Variant ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Polymorphism Analysis ◽  
Β Subunit ◽  
Wild Type ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Naturally Occurring ◽  
Single Stranded Conformational Polymorphism

Abstract The hCGβ gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGβ gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGβ gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGβ gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in β gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether βV79M hCG had biological properties that differed from those of wild-type hCG, a β-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone α-subunit in Chinese hamster ovary cells and 293T cells. When we examined βV79M hCG biosynthesis, we detected atypical βV79M hCG folding intermediates, including a βV79M conformational variant that resulted in aβ -subunit with impaired ability to assemble with the α-subunit. The inefficient assembly of βV79M hCG appeared to be independent ofβ -subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the βV79M subunit to fold correctly. The majority of the V79M β-subunit synthesized was secreted as unassembled free β. Although the amount of αβ hCG heterodimer formed and secreted by βV79M-producing cells was less than that by wild-type β-producing cells, the hCG that was secreted as αβ V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.

scholarly journals Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

2004 ◽  
Vol 279 (44) ◽  
pp. 46035-46045 ◽  
Author(s):  
Fengyu Qiao ◽  
Jun Mi ◽  
James B. Wilson ◽  
Gang Zhi ◽  
Natalie R. Bucheimer ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Mutant Cells

Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others (Qiao, F., Moss, A., and Kupfer, G. M. (2001)J. Biol. Chem. 276, 23391–23396 and Futaki, M., Watanabe, S., Kajigaya, S., and Liu, J. M. (2001)Biochem. Biophys. Res. Commun. 281, 347–351) have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in anin vitrokinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-typeFANCGcDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.

scholarly journals Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease

2020 ◽  
Vol 295 (10) ◽  
pp. 3115-3133
Author(s):  
Xiaotian Zhong ◽  
Srinath Jagarlapudi ◽  
Yan Weng ◽  
Mellisa Ly ◽  
Jason C. Rouse ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Structure Function ◽  
Fibroblast Growth Factor 23 ◽  
Chinese Hamster ◽  
Receptor Activity ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Glycan Structure

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo. Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N′-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

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