The NC16A domain of collagen XVII plays a role in triple helix assembly and stability

Bergh, Françoise Van den; Fu, Chang-Ling; Olague-Marchan, Mónica; Giudice, George J.

doi:10.1016/j.bbrc.2006.09.147

Cited by 15 publications

(13 citation statements)

References 12 publications

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“…The cDNA cloning, expression, and purification of these recombinant proteins were described previously. (5,17,18) Briefly, cDNA encoding the entire NC16A domain, or overlapping segments thereof, were obtained by PCR amplification and cloned into the pGEX-2T vector (GE Healthcare Life Sciences, Piscataway, NJ). The relative positions of the BP180 subdomains have been described (5) and are referred to as NC16A1, NC16A2, NC16A2.5, NC16A3, and NC16A1-3.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of an IgE-Class Monoclonal Antibody Specific for the Bullous Pemphigoid Autoantigen, BP180

et al. 2012

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BP180 (collagen XVII) is the target antigen in several autoimmune diseases including bullous pemphigoid (BP). Both IgE and IgG class autoantibodies have been shown to be pathogenic in BP; however, studies designed to elucidate the patho-mechanisms mediated specifically by the IgE-class autoantibodies are limited by the low levels (ng/mL) of IgE in human sera. In this report, we developed mouse IgE class monoclonal antibodies (MAbs) against the immunodominant NC16A domain of the human BP180 protein and characterized two of the resultant MAbs, designated 395A5 and 395D2. Epitope mapping studies revealed that both MAbs target segment 2 of NC16A, as was described for IgE and IgG class BP autoantibodies. Also similar to BP IgE, MAb 395A5 showed indirect immunofluorescence labeling of the basement membrane zone (BMZ) of human skin, stimulated histamine release from mast cells when triggered with NC16A, and induced keratinocyte production of IL-8. The 395D2 MAb was also able to trigger antigen-specific histamine release from mast cells; however, in contrast to BP IgE and 395A5, 395D2 did not label the cutaneous BMZ, nor did it induce IL-8 production in keratinocytes. In summary, these studies underscore the importance of functionally characterizing MAbs generated for use in human disease models. The 395A5 IgE class murine MAb was shown to share several key functional properties with the pathogenically active IgE produced by BP patients. We therefore expect that this MAb will prove to be a useful tool for dissecting the mechanisms used by BP180-NC16A-specific IgE antibodies in the induction of BP skin lesions.

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Section: Methodsmentioning

confidence: 99%

Functional Characterization of an IgE-Class Monoclonal Antibody Specific for the Bullous Pemphigoid Autoantigen, BP180

et al. 2012

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“…The expression and purification of glutathione S-transferase (GST) and the GST-NC16A fusion protein were described previously (Van den Bergh et al, 2006). Briefly, the control GST protein and GST-NC16A, consisting of the NC16A domain of human BP180 with a C-terminal collagen peptide [(Gly-Pro-Pro) 10 ] and an N-terminal glutathione S-transferase (GST) moiety, were expressed in E. coli (Rosetta strain for GST-NC16A; DH5α strain for GST) using the pGEX-2T expression system (Pharmacia Biotech, Piscataway, NJ).…”

Section: Methodsmentioning

confidence: 99%

A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid

Messingham

Noe

Chapman

et al. 2009

Journal of Immunological Methods

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Bullous pemphigoid (BP) is a humoral autoimmune disease directed predominantly against the noncollagenous NC16A domain of the BP180 hemidesmosomal protein. Our laboratory has recently shown, using a mouse xenograft model, that passive transfer of IgE autoantibodies from BP sera induces a skin phenotype that recapitulates the early phases of the disease. Herein, we describe the development of a highly specific and sensitive ELISA to detect circulating IgE autoantibodies that recognize BP180-NC16A. Using this assay, we detect a high frequency (77%) of NC16A-specific IgE class autoantibodies in the sera of BP patients. This frequency, which is significantly higher than reported previously, is comparable to that of anti-NC16A IgG autoantibody production. In 3 BP patients monitored over time, the circulating NC16A-specific levels of both IgE and IgG were associated with clinical disease activity; however, patient sera did not always contain high levels of both isotypes. In conclusion, our ELISA provides a highly sensitive and specific tool for the detection of BP180-specific IgE in patient sera. Furthermore, we report that the majority of BP sera contain both IgE and IgG class autoantibodies specific for NC16A and suggest that screening for both isotypes of autoantibodies may provide a better diagnostic value than IgG alone.

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“…Interestingly, in collagen XVII, the physiological cleavage sites are located 8 -11 amino acid residues C-terminally from the coiled coils within the NC16A domain (16), indicating the coiled coils are not included in the shed ectodomain. In addition, it has been reported that the recombinant collagenous COL15 domain of collagen XVII can form a trimeric structure without the NC16A domain (25), suggesting that trimerization of collagen XVII may not always require coiled-coil repeats. These findings led us to investigate the biological functions of coiled coils in the NC16A domain of collagen XVII.…”

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Coiled Coils Ensure the Physiological Ectodomain Shedding of Collagen XVII

Nishie

Jacków

Hofmann

et al. 2012

Journal of Biological Chemistry

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Background: ␣-Helical coiled coils represent potential oligomerization motifs in vital proteins. Results: Targeting coiled coils in the juxtamembranous linker region of transmembrane collagen XVII significantly increased intracellular cleavage by unmasking a furin recognition sequence. Conclusion: Coiled coils in collagen XVII play a pivotal role to ensure its physiological ectodomain shedding. Significance: This study revealed a novel role of coiled coils as stabilizer of a specific protease cleavage site.

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The NC16A domain of collagen XVII plays a role in triple helix assembly and stability

Cited by 15 publications

References 12 publications

Functional Characterization of an IgE-Class Monoclonal Antibody Specific for the Bullous Pemphigoid Autoantigen, BP180

Functional Characterization of an IgE-Class Monoclonal Antibody Specific for the Bullous Pemphigoid Autoantigen, BP180

A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid

Coiled Coils Ensure the Physiological Ectodomain Shedding of Collagen XVII

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