Inhibition of MEPE cleavage by Phex Rong Guo, a Peter S.N. Rowe, b Shiguang Liu, a Leigh G. Simpson, a Zhou-Sheng Xiao, a and L. Darryl Quarles a, * a Department of Medicine, The Center for Bone and Mineral Disorders, Duke University Medical Center, Box 3036, Durham, NC 27710, USA b The University of Texas Health Science Center at San Antonio, Institute for Drug Development, Molecular Therapies, Endocrinology, Texas Research Park, 14960 Omicron Drive, 78245 San Antonio, TX, USA Received 7 August 2002 Abstract X-linked hypophosphatemia (XLH) and the Hyp-mouse disease homolog are caused by inactivating mutations of Phex which results in the local accumulation of an unknown autocrine/paracrine factor in bone that inhibits mineralization of extracellular matrix. In these studies, we evaluated whether the matrix phosphoglycoprotein MEPE, which is increased in calvaria from Hyp mice, is a substrate for Phex. Using recombinant full-length Phex (rPhexWT) produced in Sf9 cells, we failed to observe Phex- dependent hydrolysis of recombinant human MEPE (rMEPE). Rather, we found that rPhex-WT inhibited cleavage of rMEPE by endogenous cathepsin-like enzyme activity present in Sf9 membrane. Sf9 membranes as well as purified cathepsin B cleaved MEPE into two major fragments of 50 and 42kDa. rPhexWT protein in Sf9 membrane fractions, co-incubation of rPhexWT and cathepsin B, and pre-treatment of Sf9 membranes with leupeptin prevented the hydrolysis of MEPE in vitro. The C-terminal domain of Phex was required for inhibition of MEPE cleavage, since the C-terminal deletion mutant rPhex (1–433) [rPhex3 0 M] failed to inhibit Sf9-dependent metabolism of MEPE. Phex-dependent inhibition of MEPE degradation, however, did not require Phex enzymatic activity, since EDTA, an inhibitor of rPhex, failed to block rPhexWT inhibition of MEPE cleavage by Sf9 membranes. Since we were unable to identify interactions of Phex with MEPE or actions of Phex to metabolize cathepsin B, Phex may be acting to interfere with the actions of other enzymes that degrade extracellular matrix proteins. Although the molecular mechanism and biological relevance of non-enzymatic actions of Phex need to be established, these findings indicate that MEPE may be involved in the pathogenesis defective mineralization due to Phex deficiency in XLH and the Hyp-mouse. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: M13 endopeptidase; Mineralization; Bone; XLH; Hyp Phex is a zinc-dependent type II cell-surface mem- brane metalloprotease that is involved in regulating phosphate and mineral homeostasis [1–4]. Inactivating mutations of Phex lead to defective calcification of bone and cartilage and renal phosphate wasting in the clinical disorder X-linked hypophosphatemic rickets (XLH) [3,5,6] and the Hyp and Gy mice disease homologs [7–9]. The pathogenesis of phosphate wasting and impaired mineralization in these disorders remain poorly under- stood, but may represent the accumulation of distinct Phex substrates or the accumulation of factors as an indirect consequence of inactivating Phex mutations. Related endopeptidases have multiple substrates that are often co-expressed with the enzyme in different tis- sues [10]. Parabiosis experiments [11] and kidney cross-trans- plantations [12] in the Hyp-mouse homolog of XLH indicated that defective Phex function leads to the ac- cumulation of a circulating phosphaturic factor, called phosphatonin [6]. Recent investigations have identified FGF23, a phosphaturic factor that is expressed by tu- mors causing oncogenic hypophosphatemic osteomala- cia (OHO) [13,14], and mutated in autosomal dominant hypophosphatemic rickets (ADH) [15], as a possible Phex substrate and candidate for phosphatonin [16,17]. Increased circulating FGF23 levels, however, do not fully explain the impaired mineralization associated with inactivation of Phex. First, inhibition of mineralization Biochemical and Biophysical Research Communications 297 (2002) 38–45 www.academicpress.com BBRC * Corresponding author. Fax: 1-919-684-4476. E-mail address: quarl001@mc.duke.edu (L. Darryl Quarles). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)02125-3