Interaction between amino propeptides of type XI procollagen alpha1 chains

Abstract

Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the alpha1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of alpha1(XI) amino propeptide (alpha1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000-50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant alpha1(XI)Npp and the endogenous alpha1(XI)Npp was observed, and specificity for alpha1(XI)Npp but not alpha2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of alpha1(XI)Npp and the endogenous alpha1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50-60 kDa on an SDS-polyacrylamide gel.

Fig.1. Expression of recombinant rat α1(XI)Npp

A, schematic representation of the amino-terminal domain of a type XI collagen molecule. The Npp of the α1(XI) chain is shown as a terminal globular domain attached to the minor helix (mh) via an extended variable region (vr). The α2(XI)Npp is absent from this schematic to represent the rapid proteolytic processing of this chain. Between the minor helix and the major triple helix (TH) lies the amino telopeptide (tp). Black bar represents location of antibody recognition site. B, extracts of E. coli cells grown in the presence of IPTG were resolved by SDS-PAGE on a 12% polyacrylamide gel stained with Coomassie Blue. Lane 1, molecular weight markers; lane 2, mid-log culture grown in the absence of IPTG; lane 3, mid-log culture grown in the presence of IPTG; lane 4, insoluble protein after extraction of E. coli cell pellet; lane 5, soluble protein after protein extraction; lane 6, flow-through from nickel-affinity column; lane 7, column wash; lanes 8 and 9, elution of purified, refolded recombinant protein; lane 10, recombinant protein markers.

Fig.2. Detection of α1(XI)Npp: α1(XI)Npp interaction by yeast two-hybrid system

A, yeast transformants plated on selective media (minus tryptophan and minus leucine) as indicated in schematic. B, single and double transformants plated in the absence of tryptophan-, leucine-, and histidine-selective medium. C, β-galactosidase activity detected by colony lift and liquid culture assay.

Fig.3. Npp:Npp interaction detected by size exclusion chromatography and light scattering

Weight average molar mass plotted as a function of elution volume from TSK 3000SW column. Buffer conditions varied from low ionic strength, 75 mm (diamonds), physiological ionic strength, 175 mm (squares), and high ionic strength, 500 mm (×). Average molecular weight values are shown for the trailing and leading edges of chromatographic peaks.

Fig.4. ELISA assay

Binding was shown to be saturable by ELISA assay in which recombinant α1(XI)Npp was bound to the plate and tagged recombinant α1(XI) amino-terminal domain was allowed to interact with the bound α1(XI)Npp. Antibody to the unique tag region was used to detect interaction. Absorbance at 405 nm was plotted as a function of tagged α1(XI) concentration (■). This antibody did not recognize α1(XI)Npp. Inset depicts the results of Scatchard analysis of binding. Bovine serum albumin was used as a control for nonspecific interaction (). Error bars indicate ± S.E.

Fig.5. Specificity of α1(XI)Npp interaction by co-precipitation of endogenous α1(XI)Npp with recombinant α1(XI)Npp-nickel-agarose

Proteins were extracted from chondrocyte pellet cultures in 1 m NaCl-containing buffer. Buffer conditions were changed by dialysis to reduce the salt concentration to 150 mm NaCl, and extract was incubated with α1(XI)Npp-bound nickel-agarose beads. Proteins were resolved by SDS-PAGE 12% polyacrylamide gel and analyzed by immunoblot using an antibody to α1(XI)Npp and an antibody to α2(XI)Npp. Lanes 1 and 3, material not bound to α1(XI)Npp-bound nickel-agarose beads. Lanes 2 and 4, material bound to α1(XI)Npp-bound nickel-agarose beads. Lanes 1 and 2, immunoblot with α2(XI)Npp antibody. Lanes 3 and 4, immunoblot with α1(XI)Npp antibody. Lane 5, molecular weight markers. Arrows on left-hand side indicate position of the α1(XI)Npp and the α2(XI)Npp.

Fig. 6. Characterization of specificity of antibody and recombinant α1(XI)Npp

Specific changes made to the recombinant α1(XI)Npp allowed the antibody to distinguish endogenous bovine α1(XI)Npp from recombinant rat α1(XI)Npp. A, recombinant α₁(XI)Npp in 10× increasing incremental concentration curve. Lane 1, 0.01-μg protein; lane 2, 0.1-μg protein; lane 3, 1-μg protein; lane 4, 10-μg protein; lane 5, molecular weight markers, 43 and 28 kDa; lane 6, 0.1-μg protein; lane 7, 1.0-μg protein; lane 8, 10-μg protein. B, nickel affinity and immunoblot of recombinant protein. Lanes 1–8, same samples as in A. Lanes 1–4, nickel affinity blot; lanes 6–8, immunoblot with α1(XI)Npp antibody. C, sequence of antigenic peptide compared with the analogous site in bovine and rat α1(XI)Npp and with the sequence of the recombinant protein. Although the antibody recognized endogenous rat α1(XI) collagen chains, it did not recognize the recombinant protein. The proteolytic processing site is indicated by the arrow.

Fig.7. Incubation of recombinant α₁(XI)Npp with bovine chondrocytes in pellet culture

A, conversion of monomer to dimer by covalent interaction between endogenous bovine α1(XI)Npp and recombinant α1(XI)Npp. Lanes 1–3, nickel affinity blot; lanes 4–7, immunoblot with antibody to endogenous α1(XI)Npp. Lane 1, purified recombinant α1(XI)Npp containing a His₆ tag migrated with an approximate size of 30 kDa on an SDS (11.5%) polyacrylamide gel after 24-h incubation at 37 °C in tissue culture medium in the absence of cells; lanes 2 and 4, no proteins were detected by nickel-NTA-alkaline phosphatase from control cell pellet cultures incubated in the absence of recombinant α1(XI)Npp for 24 h; however, endogenous α1(XI)Npp was detected; lanes 3 and 5, material extracted (in 1 m NaCl-containing buffer) from cell pellet cultures, incubated in the presence of 10 μg/ml recombinant α1(XI)Npp, contained the 30-kDa recombinant protein and some of the endogenous α1(XI)Npp (apparent molecular mass, 30 kDa) and in addition contained larger proteins including a 35-kDa protein and a protein with an apparent molecular mass of 60 kDa, which was recognized by nickel-NTA-alkaline phosphatase (lane 3) and the endogenous-specific α1(XI)Npp antibody (lane 5). Lane 6, material extracted (in 1 m NaCl-containing buffer) from cell pellet cultures incubated in the presence of 1 μg/ml recombinant α1(XI)Npp without a His₆ tag. The 60-kDa band is present as well as a minor band present at 30 kDa. Lane 7, material extracted (in 1 m NaCl-containing buffer) from cell pellet cultures incubated in the presence of 10 μg/ml recombinant α1(XI)Npp without a His₆ tag. The 60-kDa band is present as well as the 30-kDa band and a minor band, which migrates with an apparent molecular mass of 35 kDa.