Human proline specific peptidases: A comprehensive analysis

Dunaevsky, Yakov E.; Tereshchenkova, Valeriia F.; Oppert, Brenda; Belozersky, M. A.; Filippova, I. Yu.; Elpidina, Elena N.

doi:10.1016/j.bbagen.2020.129636

Cited by 32 publications

(28 citation statements)

References 188 publications

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“…Dipeptidyl peptidase 4 (DPP4), also known as CD26 and adenosine deaminase binding protein (ADAbp), is a 110 kDa type II transmembrane glycoprotein belonging to the DPP4 gene family of serine proteases. DPP4 is widely expressed on endothelial, epithelial and immune cells in mammalian tissues and has multifunctional roles in metabolism, immunology, endocrinology, fibrosis and cancer [1][2][3][4][5]. DPP4 is often cleaved from cell surfaces to be released into extracellular spaces as an enzymatically active, soluble form that has intact protein-protein binding activities [6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Xi¹,

Fazio²,

Nadvi³

et al. 2020

Preprint

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Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

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Section: Introductionmentioning

confidence: 99%

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Xi¹,

Fazio²,

Nadvi³

et al. 2020

Preprint

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“…In addition to collagen, many bioactive peptides contain proline to protect them from unexpected degradation. Prolidase and prolinase can degrade proline in these peptides to modulate the immune response, cell growth and neural development (Dunaevsky et al 2020;Misiura and Miltyk 2020). Mutations of PEPD gene can cause prolidase deficiency, a rare autosomal recessive disorder with severe skin lesions, immunodeficiency and mental retardation (Kitchener and Grunden 2012).…”

Section: Sources Of Free Prolinementioning

confidence: 99%

Proline metabolism and transport in retinal health and disease

Zhu

Lim

et al. 2021

Amino Acids

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The retina is one of the most energy-demanding tissues in the human body. Photoreceptors in the outer retina rely on nutrient support from the neighboring retinal pigment epithelium (RPE), a monolayer of epithelial cells that separate the retina and choroidal blood supply. RPE dysfunction or cell death can result in photoreceptor degeneration, leading to blindness in retinal degenerative diseases including some inherited retinal degenerations and age-related macular degeneration (AMD). In addition to having ready access to rich nutrients from blood, the RPE is also supplied with lactate from adjacent photoreceptors. Moreover, RPE can phagocytose lipid-rich outer segments for degradation and recycling on a daily basis. Recent studies show RPE cells prefer proline as a major metabolic substrate, and they are highly enriched for the proline transporter, SLC6A20. In contrast, dysfunctional or poorly differentiated RPE fails to utilize proline. RPE uses proline to fuel mitochondrial metabolism, synthesize amino acids, build the extracellular matrix, fight against oxidative stress, and sustain differentiation. Remarkably, the neural retina rarely imports proline directly, but it uptakes and utilizes intermediates and amino acids derived from proline catabolism in the RPE. Mutations of genes in proline metabolism are associated with retinal degenerative diseases, and proline supplementation is reported to improve RPE-initiated vision loss. This review will cover proline metabolism in RPE and highlight the importance of proline transport and utilization in maintaining retinal metabolism and health.

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“…The expression plasmid for soluble trimeric SARS-CoV-2 spike protein (residues 1-1208) was generously provided by Dr Florian Krammer (Icahn School of Medicine, Mt Sinai) [54,55]. The SARS-CoV-2 spike expression construct includes the proteins native signal peptide (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14] to enable secretion, proline substitutions at residues 986 and 987 for stability, a GSAS substitution at the furin cleavage site (residues 682-685), and an N-terminal His 6 -tag to allow purification. The SARS-CoV-2 receptor-binding domain (RBD) of the spike protein (residues 328-531) was cloned into the pCAGGS expression plasmid with an N-terminal IgK signal peptide, to target the protein for secretion, and a C-terminal His 9 -tag and Avitag™ to enable purification.…”

Section: Generation Of Expression Constructsmentioning

confidence: 99%

“…Dipeptidyl peptidase 4 (DPP4), also known as CD26 and adenosine deaminase binding protein (ADAbp), is a 110 kDa type II transmembrane glycoprotein belonging to the DPP4 gene family of serine proteases. DPP4 is widely expressed on endothelial, epithelial and immune cells in mammalian tissues and has multifunctional roles in metabolism, immunology, endocrinology, fibrosis and cancer [1][2][3][4][5][6]. DPP4 is often cleaved from cell surfaces to be released into extracellular spaces as an enzymatically active, soluble form that has intact protein-protein binding activities [7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Fazio

Nadvi

et al. 2020

Molecules

View full text Add to dashboard Cite

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

show abstract

Human proline specific peptidases: A comprehensive analysis

Cited by 32 publications

References 188 publications

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Proline metabolism and transport in retinal health and disease

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

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