Abstract
Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.
Highlights
Studies with recombinant nidogen showedthe presence of three globular domains, G1, G2, and G3, from the N to theC terminus, with G1 and G2 being connected by a flexible link and G2 and G3 by arod-like element consisting of five EGF’-like repeats (Foxet al., 1991)
This structure is in agreement with finity chromatography onzinc- and cobalt-loaded col- previous predictions from the cDNA-derived amino acid seumns showed binding of domains G2 and G 3 and the quences of murine and human nidogen
The high affinity ishedbyreduction andalkylation of nidogen. This binding with laminin has been localized to the G3 domain of allowed for theisolation of several zinc-binding trypticboth tissue-derived and recombinant nidogen (Mann et al, peptides, four from G2, two from the rod, and one 1f9r8o8m; Fox et al, 1991) and to a region centered around three the G3domain
Summary
All cell clones except those transfected with pNd-IIa flexible protease-sensitivelink(Fox et al, 1991), we used showed significant production of the expected protein frag- proteolysis for their separation. Exclusive binding to a cobalt-loaded chelating colamino acid residues from its N-terminal end corresponding umn of a 37-kDa fragment comprising domain G2 was demto therelatively hydrophobicbut cysteine-free subdomain IIIa onstrated by electrophoresis and sequence analysis The nonbound fraction possessed a major 30-kDa with aG3-specific antibody (Mannet al., 1988) showed a weak fragment identified as the N-terminal globe G1 by sequence band of-30 kDa in the cell extract but not in the culture analysis (Table I) together with some minor products
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
