INTRODUCTION Basement membranes are networks of interactive, multidomain proteins with high local concentrations of components. Cells interact with adjacent extracellular matrix (ECM) during cell movements. During development, organs and their adjacent basement membranes form, and enlarge, yet little is known about their mutual, evolving relationships. In a survey of proteins secreted by cultured Drosophila cells (Fessler et al., 1994) we characterized several homologues of vertebrate ECM and some novel proteins, among them papilin. It is a large glycoprotein with O-linked sulfated glycosaminoglycan chains, a propensity for disulfide-linked network formation and it occurs in basement membranes (Campbell et al., 1987). Drosophila basement membranes mature during embryonic and larval stages, as some components are added at different times. At some sites we find distinct, early deposits of papilin and other glycoproteins that never mature into basement membranes. The discovery that an ADAMTS-metalloproteinase (Kuno et al., 1997) is essential for nematode gonadogenesis highlights the possibility of repetitive cleavage, as well as synthesis, of pericellular ECM during organogenesis (Blelloch and Kimble, 1999). Papilin shares a set of protein domains, named the papilin cassette, with ADAMTS-metalloproteinases, and papilin is shown to influence organogenesis. We propose that the non-enzymatic papilin helps to focus action locally, especially of proteases, during development, turnover and repair. MATERIALS AND METHODS Papilin protein Native papilin was isolated from Drosophila Kc7E10 cell culture medium or embryos (Campbell et al., 1987), ‘core protein’ from cell cultures grown in medium devoid of sulfate and containing 10 mM NaClO3. Denatured, reduced papilin was extracted from staged embryos, larvae, pupae and adults with 2% SDS at 100°C or with guanidine HCl. Equilibrium buoyant density analyses and western blots were as described by Fessler et al. (1994). Papilin cDNA analysis For microsequencing papilin was further purified by CsCl-guanidine HCl buoyant density equilibrium centrifugation, deglycosylated with 5475 Development 127, 5475-5485 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 DEV3299 Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved ‘papilin cassette’ of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine- rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N- proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis. Key words: Papilin, Basement membrane, Drosophila melanogaster, Human, Mouse, Caenorhabditis elegans, Metalloproteinases SUMMARY Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases Irina A. Kramerova 1 , Nobuko Kawaguchi 1, *, Liselotte I. Fessler 1 , Robert E. Nelson 1 , Yali Chen 1 , Andrei A. Kramerov 1 , Marion Kusche-Gullberg 1,‡ , James M. Kramer 2 , Brian D. Ackley 2 , Aleksander L. Sieron 3 , Darwin J. Prockop 3 and John H. Fessler 1,¶ 1 MCD Biology Department and Molecular Biology Institute, University of California at Los Angeles, CA 90095, USA 2 Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA 3 Center for Gene Therapy, MCP University, Philadelphia PA19102, USA *Present address: Graduate School of Agricultural Sciences, Nagoya University, Japan ‡ Present address: Department of Medical and Physiological Chemistry, University of Uppsala, Sweden ¶ Author for correspondence (e-mail: fessler@mbi.ucla.edu) Accepted 19 September; published on WWW 14 November 2000