pubs.acs.org/Biochemistry r XXXX American Chemical Society Biochemistry XXXX, XXX, 000–000 A DOI: 10.1021/bi101187f A Novel Disulfide Pattern in Laminin-Type Epidermal Growth Factor-like (LE) Modules of Laminin β1 and γ1 Chains † Stefan Kalkhof, ‡,4,# Konstanze Witte, ‡,# Christian H. Ihling, ‡ Mathias Q. M€ uller, ‡ Manuel V. Keller, §, ) Sebastian Haehn, § Neil Smyth, ^ Mats Paulsson, §, ) and Andrea Sinz* ,‡ ‡ Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120 Halle (Saale), Germany, § Center for Biochemistry and Center for Molecular Medicine, Faculty of Medicine, University of Cologne, D-50931 Cologne, Germany, ) Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), D-50931 Cologne, Germany, and ^ School of Biological Sciences, University of Southampton, East Southampton SO16 7PX, United Kingdom 4 Present address: Department of Proteomics, Helmholtz Center for Environmental Research;UFZ, D-04318 Leipzig, Germany # Both authors contributed equally to this work Received July 27, 2010; Revised Manuscript Received August 18, 2010 ABSTRACT: In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin β1 and γ1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal β1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin γ1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns. Epidermal growth factor (EGF) 1 domains are extracellular protein modules, which are cross-linked by intramolecular di- sulfide bonds. Structurally, the classical EGF domain is described as a small domain of 30-40 amino acids primarily stabilized by three disulfides with the connectivity (1-3, 2-4, 5-6) (1). Two different types of three-disulfide EGF domains can be differ- entiated on the basis of the structure of the C-terminal half of the last disulfide loop (2). In addition to these three-disulfide EGFs, structures of three four-disulfide EGF domains, derived from the laminin γ1 chain and integrin β2 and β3 subunits, have been solved (3-5). These four-disulfide EGF domains possess an additional intradomain disulfide showing a (1-3, 2-4, 5-6, 7-8) connectivity called “laminin-type EGF-like domain” or a (1-5, 2-4, 3-6, 7-8) connectivity termed “integrin-type EGF- like domain”. Laminins are the major noncollagenous proteins within the basement membrane and are crucial for its formation. Laminin short arms are formed by the C-terminal portions of laminin chains and are required for laminin polymerization. They consist of two (β and γ) or three (R) globular domains interspersed by series of laminin-type EGF-like (LE, for nomenclature see refs 6 and 7) modules. The N-terminal (LN) globular domains are not self-folding entities but require the following four LE domains for efficient expression (8). This region of the molecule is involved in laminin polymerization and binds to the corresponding domain upon other laminin chains (9). It has also been implicated as harboring a binding site for integrin R3β1 on the laminin R5 chain (10). LE domains were originally classified as a variant of EGF modules (11, 12). Each LE domain is comprised of 50-60 amino acids, and the module was initially identified in R1, β1, and γ1 chains of laminin (11-13). LE modules are shared by all other laminin chain isoforms, where they occur in tandem ar- rays (14, 15), and by the basement membrane proteoglycans perlecan (16) and agrin (17). Series of LE domains also occur along with LN domains in netrins (18) and usherin (19), both being basement membrane associated proteins. The LE domain 8 in the laminin γ1 chain provides the high-affinity binding site for nidogen-1 (20), and it has been proposed that laminin LE domains may carry growth factor activity (21). Here we report the in-depth mass spectrometric analysis of the disulfide bond pattern in recombinant mouse laminin β1 and γ1 † This work was funded by grants SI 867/7-1 and SM 65/1-3 from the DFG (Deutsche Forschungsgemeinschaft). S.K. acknowledges support by the International Postgraduate Programme (IPP) at the University of Leipzig and the DFG-funded Graduiertenkolleg 1026 “Conformational Transitions in Macromolecular Interactions” at the Martin Luther University Halle-Wittenberg. *Address correspondence to this author. Tel: þ49-345-5525170. Fax: þ49-345-5527026. E-mail: andrea.sinz@pharmazie.uni-halle.de. 1 Abbreviations: CID, collision-induced dissociation; DTT, dithio- threitol; EGF, epidermal growth factor; EHS, Engelbreth-Holm- Swarm; ESI-FTICR-MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; HCCA, R-cyano-4-hydroxy- cinnamic acid; HCD, higher-energy collision-induced dissociation; HPLC, high-performance liquid chromatography; LE domain, laminin epidermal growth factor-like domain; LID, laser-induced dissociation; LN domain, laminin N-terminal domain (domain VI); LTQ, linear ion trap (ThermoFisher Scientific); MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PMF, peptide mass fingerprint; PFF, peptide fragment fingerprint; TGF, transforming growth factor.