BP180 Autoantibodies Target Different Epitopes in Multiple Sclerosis or Alzheimer’s Disease than in Bullous Pemphigoid

Abstract

Neurologic patients have an increased risk for bullous pemphigoid (BP), in which autoantibodies target BP180, a cutaneous basement membrane protein also expressed in the brain. Here we show that 53.6% of sera from patients with multiple sclerosis (MS) (n ¼ 56) had IgG reactivity against full-length BP180 in immunoblotting, while in BP180 non-collagenous 16A ELISA (n ¼ 143), only 7.7% of MS samples studied were positive. Epitope mapping with 13 fusion proteins covering the entire BP180 polypeptide revealed that in MS and Alzheimer's disease (AD) patients, IgG autoantibodies target regions located in the intracellular and mid-extracellular parts of BP180, but not the well-known BP epitopes located in the non-collagenous 16A domain and the distal part of extracellular domain. In indirect immunofluorescence analysis, 8.1% of MS sera recognized the cutaneous basement membrane and in full-length BP180 ELISA analysis, 7.5% MS and AD sera were positive, indicating that these autoantibodies rarely recognize BP180 in its native conformation. Thus, in MS and AD patients, BP180 autoantibodies have a different epitope profile than in patients with BP, and seldom bind to native BP180. This explains the inability of these autoantibodies to cause skin symptoms. Our results suggest that the autoantibodies against BP180 alone are not sufficient to induce BP in MS and AD patients.

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment

“…BP180 ICD (encoding aa 2-467 and 2-455) was PCR amplified from human BP180 cDNA (Franzke et al 2004), cloned into pGEX-2T (Tuusa et al 2019) or modified pET-21a vector. It was then expressed in Escherichia coli BL21(DE3) in ZYM-5052 auto-induction medium (Studier 2014) at 18 °C as Glutathione-S-transferase (GST) or N-or C-terminal 6xhistidine (His)-tagged fusion proteins.…”

“…A construct with a C-terminal His-tag was cloned by ligating oligonucleotide 5 and 6 (Table 1)-primer amplified cDNA directly into the NheI and HindIII restriction sites of pET21a. The DNA constructs encoding BP180 ICD fragments and affinity purification of the GST-fusion proteins were as described previously (Tuusa et al 2019). To purify the His-tagged BP180 ICD, cell pellets were lysed by sonication in a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 1 mM dithiotreitol, 10 mM imidazole, 100 μg ml −1 lysozyme, 10 μg ml −1 DNaseI, and cOmplete EDTA-free protease inhibitor cocktail (Merck, KGaA, Darmstadt, Germany).…”

“…The position of BP180 ICD is marked with a green diamond. d GST-BP180 fusion proteins FP1 (aa 2-168), FP2 (147-305), FP3 (261-401) and FP4 (377-455) were expressed in Escherichia coli and glutathione sepharose pull downs (Tuusa et al 2019) were analyzed by immunoblotting with anti-GST antibody. In the case of FP1 and FP2, the polypeptide of highest molecular weight represents the full-length fusion protein.…”

Gliptin-associated Bullous Pemphigoid and the Expression of Dipeptidyl Peptidase-4/CD26 in Bullous Pemphigoid

“…The BP180-NC16A ELISA assay was performed in the HUSLAB, (Helsinki, Finland). Based on the observations from our previous publication (29), two glutathione-S-transferase fusion proteins, FP4 and FP5, which correspond to human BP180 amino acids 377-455 and 489-567, respectively, were immunoblotted with a subset of BP sera (n = 24) as previously described (29).…”

Recent Advances in Understanding Pemphigus and Bullous Pemphigoid

“…However, only about 8% of the MS sera reacted against the major BP180 disease epitope, BP180-NC16A, by ELISA, or against the cutaneous membrane zone by indirect immunofluorescence. These studies indicate that epitopes differ between the BP and MS anti-BP180 sera, suggesting that epitope spreading may be necessary to initiate BP (Tuusa et al, 2019).…”