Recombinant Procollagen II: Deletion of D Period Segments Identifies Sequences That Are Required for Helix Stabilization and Generates a Temperature-sensitive N-Proteinase Cleavage Site

Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. To obtain information on the conformation and the functions of this unusual collagen, its largest collagenous domain, Col15, was expressed in a eukaryotic episomal expression system and purified by DEAE and fast protein liquid-Mono S chromatography. The protein was triple-helical (T m of 26.5°C) when produced in cultures containing ascorbic acid. When the vitamin supply was limited, the 4-hydroxyproline content was reduced from 74 to 9%, which, in turn, resulted in a drastic reduction of the stability of the triple helix. The glycine substitution mutation G627V associated with junctional epidermolysis bullosa, a human blistering skin disease, also had a striking effect on thermal stability of rCol15 causing partial unfolding already at 4°C. Col15 promoted cell adhesion of epithelial and fibroblastic cell lines with a ␤1 integrinmediated mechanism. In concert with this, in acquired autoimmune blistering skin diseases, circulating IgG and IgA autoantibodies were found to target rCol15r.Collagens are a family of closely related, although genetically distinct, extracellular matrix proteins. Each collagen consists of three polypeptide chains, ␣ chains, which contain a characteristic repeating "collagenous" triplet amino acid sequence -Gly-Xaa-Yaa-, where Xaa and Yaa denote amino acids other than glycine. In all collagen types, the ␣ chains also have non-collagenous domains of varying sizes (1). Typically, the three polypeptide chains are twisted around each other into a collagen triple helix; however, only suggestive data for this exist in the recently characterized transmembrane collagens, types XIII and XVII (for review, see Ref.2). Collagen XVII, also known as the 180-kDa bullous pemphigoid antigen, or BP180, is a structural component of hemidesmosomes, multiprotein complexes that mediate the adhesion of epidermal keratinocytes to the underlying basement membrane (3, 4). The cDNA sequence codes for a type II integral transmembrane protein of 1497 amino acids, with an intracellular domain of 560 amino acids, a short transmembrane stretch, and an extracellular collagenous domain of 914 amino acids with multiple interruptions. The length of the individual collagenous subdomains varies from 14 to 242 amino acid residues (5). Recently, it was established that collagen XVII exists as two molecular forms, i.e. as a full-length transmembrane homotrimer of three 180-kDa ␣1(XVII) chains and as a 120-kDa soluble form that corresponds to the extracellular domain and is presumably released from the cell surface through furin-mediated proteolytic processing (6, 7). In some situations also a shorter, approximately 90 -100-kDa fragment, has been observed (3, 4, 6). Very little is known about the molecular shape of collagen XVII under physiological conditions. Rotary shadowing ele...

Section: Discussionmentioning

confidence: 99%

Collagen XVII Is Destabilized by a Glycine Substitution Mutation in the Cell Adhesion Domain Col15

Tasanen

Eble

Aumailley

et al. 2000

“…Medium was buffered with 100 mM Tris-HCl, pH 7.4, and cooled to 4°C. Protease inhibitors were added to obtain the following final concentrations: 250 mM EDTA, 0.2% NaN 3 , 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, and 10 mM N-ethylmaleimide (30). Procollagen was precipitated by gradual addi-tion of ammonium sulfate to a final concentration of 176 mg/ml, incubation at 4°C overnight, then centrifugation at 12,000 ϫ g for 2 h.…”

Section: Methodsmentioning

confidence: 99%

Procollagen with Skipping of α1(I) Exon 41 Has Lower Binding Affinity for α1(I) C-telopeptide, Impaired in Vitro Fibrillogenesis, and Altered Fibril Morphology

Cabral

Fertala

Green

et al. 2002

Self Cite

Previous in vitro data on type I collagen self-assembly into fibrils suggested that the amino acid 776 -796 region of the ␣1(I) chain is crucial for fibril formation because it serves as the recognition site for the telopeptide of a docking collagen monomer. We used a natural collagen mutation with a deletion of amino acids 766 -801 to confirm the importance of this region for collagen fibril formation. The proband has type III osteogenesis imperfecta and is heterozygous for a COL1A1 IVS 41 A ؉4 3 C substitution. The intronic mutation causes splicing of exon 41, confirmed by sequencing of normal and shorter reverse transcriptase-PCR products. Reverse transcriptase-PCR using RNA from proband dermal fibroblasts and clonal cell lines showed the mutant cDNA was about 15% of total ␣1(I) cDNA. The mutant transcript is translated; structurally abnormal ␣ chains are demonstrated in the cell layer of proband fibroblasts by SDSurea-PAGE. The proportion of mutant chains in the secreted procollagen was determined to be 10% by resistance to digestion with MMP-1, since chains lacking exon 41 are missing the vertebral collagenase cleavage site. Secreted proband collagen was used for analysis of kinetics of binding of ␣1(I) C-telopeptide using an optical biosensor. Telopeptide had slower association and faster dissociation from proband than from normal collagen. Purified proband pC-collagen was used to study fibril formation. The presence of the mutant molecules decreases the rate of fibril formation. The fibrils formed in the presence of 10 -15% mutant molecules have strikingly increased length compared with normal collagen, but are well organized, as demonstrated by D-periodicity. These results suggest that some collagen molecules containing the mutant chain are incorporated into fibrils and that the absence of the telopeptide binding region from even a small portion of the monomers interferes with fibril growth. Both abnormal fibrils and slower remodeling may contribute to the severe phenotype.Type I collagen is the predominant protein in the extracellular matrix of skin, bone, and tendon. In tissue, the type I collagen heterotrimer is organized into heterotypic fibrils along with types III and V collagen and non-collagenous proteins of matrix, such as decorin (1-4). Collagen fibrils in a wide range of different tissues have distinct diameter distributions (5), supporting a process for strict control over fibril growth and assembly. Structural abnormalities of type I collagen are the cause of significant medical disorders such as osteogenesis imperfecta, a genetic disorder of connective tissue characterized by bone fragility, and two of the types of Ehlers-Danlos

“…Based on accumulated structural knowledge and analysis of the amino acid sequence, we proposed that the long suspected (7)(8)(9) domain organization of the collagen triple helix plays a crucial role in ␣1(I)-OI/EDS (4). Specifically, we postulated that a distinct, highly stable "N-anchor" folding domain is formed by the first 85 N-terminal amino acids of each chain of the type I collagen triple helix.…”

mentioning

confidence: 99%

Molecular Mechanism of α1(I)-Osteogenesis Imperfecta/Ehlers-Danlos Syndrome

Makareeva

Cabral

Marini

et al. 2006

We demonstrate that 85 N-terminal amino acids of the ␣1(I) chain participate in a highly stable folding domain, acting as the stabilizing anchor for the amino end of the type I collagen triple helix. This anchor region is bordered by a microunfolding region, 15 amino acids in each chain, which include no proline or hydroxyproline residues and contain a chymotrypsin cleavage site. Glycine substitutions and amino acid deletions within the N-anchor domain induce its reversible unfolding above 34°C. The overall triple helix denaturation temperature is reduced by 5-6°C, similar to complete N-anchor removal. N-propeptide partially restores the stability of mutant procollagen but not sufficiently to prevent N-anchor unfolding and a conformational change at the N-propeptide cleavage site. The ensuing failure of N-proteinase to cleave at the misfolded site leads to incorporation of pN-collagen into fibrils. Similar, but weaker, effects are caused by G88E substitution in the adjacent triplet, which appears to alter N-anchor structure as well. As in Ehlers-Danlos syndrome (EDS) VIIA/B, fibrils containing pNcollagen are thinner and weaker causing EDS-like laxity of large and small joints and paraspinal ligaments. However, distinct structural consequences of N-anchor destabilization result in a distinct ␣1(I)-osteogenesis imperfecta (OI)/EDS phenotype.Mature type I collagen is a heterotrimer of two ␣1(I) and one ␣2(I) chains folded into a 300-nm-long triple helix with short, unstructured telopeptides at the N-and C-terminal ends. The distinguishing feature of the collagen triple helix is an obligatory Gly residue in every third position on each chain. Collagen precursor (procollagen) is synthesized and folded within the rough endoplasmic reticulum. It is secreted by cells with N-and C-terminal propeptides still attached on each side of the molecule. Subsequent cleavage of the propeptides by specialized Nand C-proteinases triggers self-assembly of the mature collagen into fibrils. The fibrils co-assemble with a variety of different extracellular matrix molecules into the structural scaffold of skin, bone, and other connective tissues. Most mutations disrupting the structure of the type I collagen triple helix (usually substitutions of one of the obligatory Gly residues) cause moderately severe to lethal forms of osteogenesis imperfecta (OI), 2 which is a clinically heterogeneous group of disorders characterized by bone fragility and skeletal deformity (1-3).In our previous study (4) we described a distinct group of ␣1(I)-OI/ EDS patients with combined clinical symptoms of the type III or IV OI as well as laxity of large and small joints and paraspinal ligaments more characteristic of Ehlers-Danlos syndrome (EDS). All of them had structural mutations within the first 90 N-terminal residues of the helical region of the ␣1(I) chain (five Gly substitutions and a 15-aa deletion). Their mutations altered procollagen structure within the N-propeptide cleavage site, resulting in incorporation of pN-collagen with uncleaved N-propeptid...