Analysis of recombinant Phex: An endopeptidase in search of a substrate

Rong Guo, Shiguang Liu, Robert F. Spurney, Leigh Quarles

Research output: Contribution to journalArticle

77 Citations (Scopus)

Abstract

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Endocrinology and Metabolism
Volume281
Issue number4 44-4
StatePublished - Oct 20 2001
Externally publishedYes

Fingerprint

Familial Hypophosphatemic Rickets
Endopeptidases
Mutant Proteins
Parathyroid Hormone
Phosphates
Thermolysin
Leucine Enkephalin
Neprilysin
Membranes
Caseins
Osteoblasts
Edetic Acid
Catalytic Domain
Western Blotting
Amino Acids
Peptides
Mutation
Proteins

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Physiology
  • Physiology (medical)

Cite this

Analysis of recombinant Phex : An endopeptidase in search of a substrate. / Guo, Rong; Liu, Shiguang; Spurney, Robert F.; Quarles, Leigh.

In: American Journal of Physiology - Endocrinology and Metabolism, Vol. 281, No. 4 44-4, 20.10.2001.

Research output: Contribution to journalArticle

@article{27fa5ad4c14745468d1f8aed51551562,
title = "Analysis of recombinant Phex: An endopeptidase in search of a substrate",
abstract = "X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.",
author = "Rong Guo and Shiguang Liu and Spurney, {Robert F.} and Leigh Quarles",
year = "2001",
month = "10",
day = "20",
language = "English (US)",
volume = "281",
journal = "American Journal of Physiology",
issn = "0193-1849",
publisher = "American Physiological Society",
number = "4 44-4",

}

TY - JOUR

T1 - Analysis of recombinant Phex

T2 - An endopeptidase in search of a substrate

AU - Guo, Rong

AU - Liu, Shiguang

AU - Spurney, Robert F.

AU - Quarles, Leigh

PY - 2001/10/20

Y1 - 2001/10/20

N2 - X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

AB - X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

UR - http://www.scopus.com/inward/record.url?scp=0034786083&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034786083&partnerID=8YFLogxK

M3 - Article

C2 - 11551862

AN - SCOPUS:0034786083

VL - 281

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0193-1849

IS - 4 44-4

ER -