FIGURES AND TABLES

Figure 1.

Schematic showing the pRC/3(IV) construct. The full-length 3(IV) cDNA was generated by polymerase chain reaction (PCR) and subcloned into Nhe I and Cla I restriction sites in the pRC-X expression vector, which was modified by adding BM40 signal peptide, FLAG peptide, and additional restriction sites.

Figure 2.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of purified r-3(IV) chain. The r-3(IV) chain was purified from the culture medium by using FLAG-affinity chromatography. Purified samples were run on 4 to 15% gradient gels under nonreducing (A) and reducing (B) conditions and were stained with Coomassie Blue. Without reduction, the r-3(IV) chain migrated as a double band with M_r> 200,000. After reduction with 20 mM dithiothreitol, the chain appeared as a single band, M_r approximately 250,000.

Figure 3.

Western blot analysis of the full-length and collagenase-digested r-3(IV) chain. The r-3(IV) chain and its collagenase digestion products were run on 4 to 15% gradient gels and were blotted with monoclonal antibody against 3(IV)NC1 (A) and Goodpasture antibodies (GP-1; B). Lane 1, molecular weight standard (Gibco); lane 2, r-3(IV) chain; lane 3, collagenase-digested r-3(IV) chain; and lane 4, r-3(IV)NC1.

Figure 4.

Rotary shadowing electron microscopy of the r-3(IV) chain. The r-3(IV) chain exists mainly in nontriple-helical form, and only the globular NC1 domains were visible by electron microscopy (A and B). In a few cases, rod-like extensions from the globular domains, representing triple-helical domains, were observed (C and D).

Figure 5.

Assessment of the binding capacity of Goodpasture (GP) antibodies to r-3(IV) chain relative to r-3(IV)NC1 by competitive enzyme-linked immunosorbent assay. The r-3(IV) chain (A and C) and the r-3(IV)NC1 (B and D) were coated on the plates. GP antibodies from one patient (GP-1) were preincubated with various concentrations of the r-3(IV) chain and the r-3(IV)NC1 before adding them to the coated wells (A and B), and five GP sera were preincubated with the r-3(IV) chain and r-3(IV)NC1 at a concentration of 80 nM before adding them to the coated wells (C and D). r-2(IV)NC1 and albumin were used as controls. Color development was measured at 410 nM.

Figure 6.

The assessment of binding capacity of Goodpasture (GP) antibodies to the r-3(IV) chain and the r-3(IV)NC1 domain by binding of GP antibodies by r-3(IV)NC1 affinity column. The r-3(IV) chain (A and C) and r-3(IV)NC1 (B and D) were coated on the plate. GP antibodies (GP-1), which bound to the r-3(IV)NC1 affinity column (A and B) and unbound antibodies (C and D) were preincubated with various concentrations of r-3(IV) chain, r-3(IV)NC1, r-2(IV)NC1, and albumin before adding them to the coated wells. Color development was measured at 410 nM.

Table 1 - Table 1 Primers used in PCR amplification reactions.

Table 2 - Table 2 Amino acid changes compared to the published primary structure of 3(IV) chain⁵ .