Expression of the human activin type I and II receptor extracellular domains in Pichia pastoris

Daly, Rachel; Hearn, Milton Thomas William

doi:10.1016/j.pep.2005.10.001

Cited by 19 publications

(9 citation statements)

References 36 publications

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“…Fractions eluted with 250 mM imidazole showed a band of w23 kDa along with a broad smearing band above the 23 kDa band. The smearing pattern was also observed previously during human, pig, chicken, and goldfish ACVR2B-ECD expression in P. pastoris (Daly & Hearn 2006, Carpio et al 2009, Kim et al 2012.…”

Section: Expression and Characterization Of Saacvr2b-1-ecdsupporting

confidence: 83%

“…Essentially similar results regarding N-glycosylation were reported for the pig ACVR2B-ECD expressed in P. pastoris (Kim et al 2012). By contrast, ACVR2B-ECD from goldfish, chicken, and human produced in P. pastoris (Daly & Hearn 2006, Carpio et al 2009, Kim et al 2012 and mouse ACVR2B-ECD produced in baculovirus-insect cell culture system (Donaldson et al 1999) also seem to undergo O-linked glycosylation (on serine or threonine residues).…”

supporting

confidence: 76%

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Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish

Funkenstein

Krol

Esterin

et al. 2012

Journal of Molecular Endocrinology

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Myostatin (MSTN), a negative regulator of muscle growth and a member of the transforming growth factor-b superfamily, can bind the two activin type 2 receptors (ACVR2). It has been previously shown that WT mice injected with ACVR2B extracellular domain (ACVR2B-ECD) had higher muscle mass. Likewise, fish larvae immersed in Pichia pastoris culture supernatant, containing goldfish Acvr2b-ECD, showed enhanced larval growth. However, it is not clear whether fish Mstn1 and Mstn2 signal through the same receptor and whether fish express more than one acvr2b gene. In the current study, three cDNAs encoding acvr2b (saacvr2b-1, saacvr2b-2a, and saacvr2b-2b) were cloned from gilthead sea bream. All three contain the short extracellular binding domain, a short transmembrane region, and a conserved catalytic domain of serine/threonine protein kinase. Bioinformatics analysis provided evidence for the existence of two acvr2b genes (acvr2b-1 and acvr2b-2) in several other fish species as well, probably as a result of gene or genome duplication. The two isoforms differ in their amino acid sequences. The direct inhibitory effect of Acvr2b-ECD on Mstn activity was tested in vitro. The saAcvr2b-1-ECD was expressed in the yeast P. pastoris. Evidence is provided for N-glycosylation of Acvr2b-1-ECD. The affinity-purified Acvr2b-1-ECD inhibited recombinant mouse/rat/human mature MSTN activity when determined in vitro using the CAGA-luciferase assay in A204 cells. A lower inhibitory activity was obtained when unprocessed purified, furin-digested, and activated saMstn1 was used. Results of this study demonstrate for the first time the existence of two acvr2b genes in fish. In addition, the study shows that bioactive fish Acvr2b-ECD can be produced from P. pastoris.

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Section: Expression and Characterization Of Saacvr2b-1-ecdsupporting

confidence: 83%

supporting

confidence: 76%

Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish

Funkenstein

Krol

Esterin

et al. 2012

Journal of Molecular Endocrinology

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“…When BMPR2 is reduced or absent, increased BMP utilization of ACVR2A/B occurs at the expense of activin signaling. Although unexpected, this finding is supported by the fact that, in general, the affinity of BMPs for ACVR2A/B is in the same range as the affinity for BMPR2 (Allendorph et al, 2006;Berasi et al, 2011;Daly and Hearn, 2006;Greenwald et al, 2003;Greenwald et al, 2004;Heinecke et al, 2009;Hu et al, 2010;Isaacs et al, 2010;Kirsch et al, 2000;Knaus and Sebald, 2001;Koncarevic et al, 2010;Rosenzweig et al, 1995;Sako et al, 2010;Sengle et al, 2008), and BMPs also possess a flexible mode of receptor complex assembly, which might enhance their ability to compete with activins that have only a single mode of complex assembly (Hinck, 2012). Because bone cells have abundant type 1 BMP receptors (ALK2, ALK3 and ALK6, BioGPS Database), preformed receptor complexes might also be biased toward BMP binding.…”

Section: Discussionmentioning

confidence: 87%

Loss of BMPR2 leads to high bone mass due to increased osteoblast activity

Lowery

Intini

Gamer

et al. 2015

Journal of Cell Science

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Imbalances in the ratio of bone morphogenetic protein (BMP) versus activin and TGFb signaling are increasingly associated with human diseases yet the mechanisms mediating this relationship remain unclear. The type 2 receptors ACVR2A and ACVR2B bind BMPs and activins but the type 2 receptor BMPR2 only binds BMPs, suggesting that type 2 receptor utilization might play a role in mediating the interaction of these pathways. We tested this hypothesis in the mouse skeleton, where bone mass is reciprocally regulated by BMP signaling and activin and TGFb signaling. We found that deleting Bmpr2 in mouse skeletal progenitor cells (Bmpr2-cKO mice) selectively impaired activin signaling but had no effect on BMP signaling, resulting in an increased bone formation rate and high bone mass. Additionally, activin sequestration had no effect on bone mass in Bmpr2-cKO mice but increased bone mass in wild-type mice. Our findings suggest a novel model whereby BMPR2 availability alleviates receptor-level competition between BMPs and activins and where utilization of ACVR2A and ACVR2B by BMPs comes at the expense of activins. As BMP and activin pathway modulation are of current therapeutic interest, our findings provide important mechanistic insight into the relationship between these pathways in human health.

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“…This supposition is supported by our experiments using the soluble ACVR2B-ECD, which revealed that BMP2 binding to native and de-glycosylated ACVR2B-ECD is identical. Comparable results were reported regarding the impact of N-glycosylation on the interaction between ACVR2A and Activin A [34,36] and Inhibin [36]. …”

Section: Discussionmentioning

confidence: 90%

N-linked glycosylation of the bone morphogenetic protein receptor type 2 (BMPR2) enhances ligand binding

Lowery

Amich

Andonian

et al. 2013

Cell. Mol. Life Sci.

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The Bone Morphogenetic Protein (BMP) signaling pathway is essential for normal development and tissue homeostasis. BMP signal transduction occurs when ligands interact with a complex of type 1 and type 2 receptors to activate downstream transcription factors. It is well established that a single BMP receptor may bind multiple BMP ligands with varying affinity, and this has been largely attributed to conformation at the amino acid level. However, all three type 2 BMP receptors (BMPR2, ACVR2A/B) contain consensus N-glycosylation sites in their extracellular domains (ECDs), which could play a role in modulating interaction with ligand. Here, we show a differential pattern of N-glycosylation between BMPR2 and ACVR2A/B. Site-directed mutagenesis reveals that BMPR2 is uniquely glycosylated near its ligand binding domain and at a position that is mutated in patients with Heritable Pulmonary Arterial Hypertension. We further demonstrate using a cell-free pulldown assay that N-glycosylation of the BMPR2-ECD enhances its ability to bind BMP2 ligand but has no impact on binding by the closely-related ACVR2B. Our results illuminate a novel aspect of BMP signaling pathway mechanics and demonstrate a functional difference resulting from post-translational modification of type 2 BMP receptors. Additionally, since BMPR2 is required for several aspects of normal development and defects in its function are strongly implicated in human disease, our findings are likely relevant in several biological contexts in normal and abnormal human physiology.

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Expression of the human activin type I and II receptor extracellular domains in Pichia pastoris

Cited by 19 publications

References 36 publications

Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish

Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish

Loss of BMPR2 leads to high bone mass due to increased osteoblast activity

N-linked glycosylation of the bone morphogenetic protein receptor type 2 (BMPR2) enhances ligand binding

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