Subject(s)
Autoantigens/chemistry , Immunoglobulin G/chemistry , Lichen Planus, Oral/immunology , Non-Fibrillar Collagens/chemistry , Administration, Oral , Biopsy , CD8-Positive T-Lymphocytes/metabolism , Enzyme-Linked Immunosorbent Assay , Epidermis/metabolism , Humans , Immunoblotting , Immunoglobulin G/metabolism , Keratinocytes/cytology , Lichen Planus, Oral/metabolism , Lymphocytes/metabolism , Microscopy, Fluorescence , Recombinant Proteins/chemistry , Collagen Type XVIIABSTRACT
Mutations in the gene COL17A1 cause non-Herlitz junctional epidermolysis bullosa. Here, we describe a patient who, despite two heterozygous mutations in COL17A1, has an extremely mild form of the disease missing most of the characteristic clinical features. DNA analysis revealed a frame-shift mutation 3432delT and a nonsense mutation 2356C-->T (Q751X). cDNA analysis showed that the deleterious effect of the latter mutation was skirted by deleting the premature termination codon containing exon 30. In this way, the reading frame was restored, resulting in a 36 nucleotides shorter mRNA transcript. Immunoblot analysis showed expression of the 180-kDa bullous pemphigoid antigen (BP180) with a slightly higher SDS-PAGE mobility, in line with the deletion of 12 amino acids from the COL15 domain. Immunofluorescence of skin sections showed diminished, but correctly localised expression of BP180, and this, in concert with the mild clinical phenotype, suggests that this COL15 mutated BP180 is still partly functional.
Subject(s)
Autoantigens/genetics , Collagen/genetics , Epidermolysis Bullosa/genetics , Frameshift Mutation/genetics , Pemphigoid, Bullous/genetics , Base Sequence , Carrier Proteins , Cytoskeletal Proteins , Dystonin , Epidermolysis Bullosa/classification , Exons , Humans , Nerve Tissue Proteins , Non-Fibrillar Collagens , Pemphigoid, Bullous/classification , RNA, Messenger/genetics , Sequence Deletion , Skin/pathology , Transcription, Genetic , Collagen Type XVIIABSTRACT
Caterpillar spicule venoms were extracted and studied for the following activities: arginine ester (BAEE) hydrolase, tyrosine ester (ATEE) hydrolase, protease (casein digestion) and phospholipase A (indirect hemolytic activity). Crude spicule venom of E. chrysorrhoea preferably hydrolyzed BAEE in contrast to E. subflava venom, which hydrolyzed ATEE in preference to BAEE. This difference was confirmed by Sephadex G-100 elution profiles. The esterase activity in E. chrysorrhoea venom was separated into two peaks with average mol. wts. of 96,000 and 44,000. The first peak demonstrated optimal BAEE hydrolysis at pH 8.6 and 37 degrees C, whereas the second peak optimally hydrolyzed both BAEE and ATEE at pH 8.45 and pH 8.6 at 45 degrees C respectively. The esterase activity in E. subflava venom was separated into two peaks with average mol. wts of 63,000 and 32,000 showing optimal hydrolysis of BAEE at pH 8.9 and 37 degrees C, and of ATEE at pH 7.75 and pH 8.5 at 40 degrees C. The column fractions showed comparable proteolytic activity, irrespective of differences between their esterase activities. The presence of phospholipase A (PLA) enzyme in crude spicule venom of both species was evident from their indirect hemolytic activities. The PLA activity eluted with the void volume and seems to be associated with some high molecular weight protein. Under the assay conditions used, E. subflava venom contained 50-100 times less PLA activity than E. chrysorrhoea venom.
Subject(s)
Arthropod Venoms/isolation & purification , Lepidoptera , Moths , Animals , Carboxylic Ester Hydrolases/isolation & purification , Hydrogen-Ion Concentration , Larva , Molecular Weight , Peptide Hydrolases/isolation & purification , Phospholipases A/isolation & purification , Species Specificity , TemperatureABSTRACT
The spicule venoms of Euproctis chrysorrhoea and Euproctis subflava were investigated for their capacity to hydrolyze chromogenic tripeptide substrates with selective affinities for various serine proteases. Seven substrates were assayed with affinities for trypsin and thrombin, trypsin and urokinase, serine proteases, chymotrypsin, glandular kallikrein, plasma kallikrein and plasmin. Venom material has a broad spectrum of affinities for the substrates with relative high plasma kallikrein activities. In E. chrysorrhoea venom, trypsin-like activities predominated, whereas E. subflava venom hydrolyzed, in preference, substrates with an affinity for chymotrypsin. The venoms were fractionated on Sephadex G-100, leading to three fractions, all having serine protease activity. The ratios of substrate specificities were markedly different, indicating that in both caterpillar venom preparations at least two separate serine proteases are present. In addition, in human plasma, inhibitor activity could be detected to the kallikrein activity of E. chrysorrhoea, but not of E. subflava. The trypsin-like activity was not inhibited by human plasma. These and earlier studies warrant the assumption that serine proteases, particularly kallikrein, are major factors in the elicitation of clinical symptoms observed after contact with caterpillar spicules.
