cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components

Abstract

Procollagen N-proteinase (EC 3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers-Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor beta or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.

Figure 1

(Upper) The oligonucleotide sequences deduced from two previously determined amino acid sequences (20). Unambiguously determined amino acids are in capital letters. (Lower) Three overlapping cDNAs provided a 4,580-nucleotide sequence containing a full-length ORF starting with an ATG codon (boldface type) in a perfectly conserved Kozak consensus sequence and ending with a TAA stop codon (boldface type) that is the beginning of an AATAAA polyadenylylation signal (italics).

Figure 2

Northern blot analysis of bovine skin RNA using pNPI specific probes. Twenty micrograms of poly(A)⁺ RNA (lanes A and B) or 40 μg of total RNA (lanes C and D) were separated by electrophoresis in agarose/formaldehyde gel, transferred onto a nylon membrane, and hybridized with pNPI-specific probes (lanes A and B, OP1 and OP10 oligonucleotide probes, respectively; lanes C and D, cDNA probes from clones 12 and 41). The membranes were exposed to x-ray film. Positions of 28S and 18S ribosomal RNA are indicated.

Figure 3

Enzymatic activity of the recombinant bovine pNPI. Recombinant enzyme recovered from the conditioned medium of the two different HT-1080 clones expressing high level of pNPI mRNA were incubated with ¹⁴C-labeled procollagen substrate in the standard reaction buffer (16). After 4 h at 35°C, reaction products were separated by SDS/PAGE and visualized after autoradiography. bpNP1–2 and bpNP1–4, Enzyme from two different clones expressing a full-length cDNA for pNPI; Proγ chains, disulfide-linked proα chains of type I procollagen; pCα1 and pCα2, partially processed proα1(I) and proα2(I) chains of type I procollagen containing the C-propeptides but not the N-propeptides from proα1(I) and proα2(I) chains. N1 and N2, N-propeptides from proα1(I) and proα2(I) chains.

Figure 4

Deduced amino acid sequence of pNPI. The 1205-amino acid protein contains a signal peptide region, composed mainly of leucine and alanine residues. Potential cleavage sites by mammalian subtilisins [RTRR (77–80)] and [RRRMRR (248,253)] and a RGD sequence (685–687) are indicated in boldface type. Potential glycosylation sites are underlined. The Zn²⁺-binding site, Met-turn, the potential transmembrane domain, and properdin repeats are indicated (below sequence).

Figure 5

Sequence comparisons. (Upper) The sequence of the Zn²⁺-binding domain of the pNPI is compared with sequences found in members of families M10 and M12 of the clan MB of metallopeptidases. Residues in boldface type indicate amino acids that are critical for Zn²⁺-binding and that represent the characteristic signature of the clan MB. (Lower) The four properdin repeats found in pNPI are compared with the three repeats present in human thrombospondin 1 (TSP1) and to four of the six repeats found in human properdin (PROP). The conserved residues are shaded. Subdomains of the second repeat in TSP1 that have previously been shown (–29) to display biological activities and homologous sequences in other repeats are boxed.

Figure 6

Analysis of pNPI expression. (A) pNPI enzymatic activity was measured in extracts of various organs and expressed in arbitrary units. (B) pNPI mRNA steady-state level was measured by RT-PCR using two different pairs of [α³²-P]-labeled primers, one allowing the amplification of part of the properdin repeat domain (A29-A30, ▪) and the other allowing the amplification of a region including the Zn²⁺-binding site (A46-A47, ░⃞). PCR-amplified products were separated on 10% polyacrylamide gels and assayed by autoradiography. Results obtained by quantification of the specific pNPI band by LASER scanning densitometry are expressed in arbitrary units. In, intestine; Lu, lung; He, heart; Sp, spleen; Ki, kidney; Br, brain; Li, liver; Ao, aorta; Bo, bone; Sk, skin; Te, tendon; Bl, bladder; Th, thymus; Re, retina; Mu, muscle.