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Molecular and Proteomic Characterization of Bla g 2 Allergen in the German Cockroach (Blattella germanica L.)
thesisposted on 17.12.2021, 16:21 authored by Aaron RodriquesAaron Rodriques
The German cockroach, Blattella germanica, is a common insect pest in the United States that causes aesthetically unpleasing collections of feces on household surfaces including walls, floors and furniture (Nalyanya et al. 2009, Pomés et al. 2002). The German cockroach is economically and medically significant not only because it contaminates food with feces, but also because the German cockroach is a physical and mechanical carrier of pathogenic bacteria, viruses and intestinal parasites (Hamu et al. 2014). In addition to being a vector, the German cockroach also produces allergenic “Bla g” proteins in multiple tissues and in its feces, including the inactive aspartic protease Bla g 2 (Arruda et al. 1995, Pomés et al. 2002). Bla g 2 has been documented by Arruda et al. (1995) to exist in several German cockroach tissues, however the abundance of Bla g 2 in adult male-exclusive structures known German cockroach tergal glands was not known. Tergal glands are structures found in numerous insect species that may contain proteins, sugars, and pheromones, and German cockroach tergal glands secrete a feeding stimulant that is consumed during courtship as well as outside of the context of courtship by conspecifics of differing sexes and life stages. Because of this wanted to assess if Bla g 2 was a constituent of the tergal glands. Furthermore, although Bla g 2 protein expression has been detected in several cockroach tissues, studies that link protein expression in various life stages and tissues with mRNA and protein abundance profiles have not been conducted. The goals of my dissertation were to measure the relative abundances of Bla g 2 mRNA and protein in different tissues and life stages, as well as identify all the proteins in the tergal glands, including the proteins that interact with Bla g 2. The molecular tools used to complete these objectives included RT-qPCR, Western blotting/ immunoblotting, co-immunoprecipitation, and liquid chromatography-tandem mass spectrometry (LC-MS). I found that Bla g 2 protein was detected in each tissue analyzed, including the tergal glands, and that protein expression was relatively high in adult males and their tergal glands in comparison to nymphs and females. Bla g 2 mRNA transcript levels were also relatively high in male tergal glands and adult males. My proteomic analysis detected three isoforms of Bla g 2 in the tergal gland secretions.
Detection of multiple isoforms in the tergal gland secretions prompted me to perform LC-MS peptide sequencing with entire tergal glands, and I was able to identify not only Bla 2 isoforms, but other protein isoforms present in the glands. Because isoforms are proteins with similar peptide sequences that originate from the same gene or gene family but typically have different structures and functions, assessing the identities and abundances of allergen isoforms that may reside in the tergal glands helped to inform me of the medical significance of the tergal glands. I was able to detect 608 proteins in the tergal glands, and LC-MS of the tergal gland secretions alone detected 303 proteins. In the co-immunoprecipitation experiments, I was also able to detect proteins that exclusively co-immunoprecipitate with Bla g 2 in the tergal glands, despite being present in the male and gravid female whole bodies as well.
I used TargetP-2.0, a bioinformatics prediction server, to identify which proteins in the tergal gland secretions were actually transported from tissue specific cells to secretions due to the presence of a signal peptide. This method was used to filter out proteins that may have contaminated the secretions from external sources, such as the wings or feces, and to filter out proteins that originated from damaged or apoptotic tergal gland cells. I also used Blast2GO, bioinformatics software that calculates the functional and locational annotations of proteins, to annotate the proteins that were profiled in the tergal glands. The data show that proteolysis is the most common biological process in the tergal gland secretion proteins that were predicted to have a signal peptide. I also found that translation is the most common biological process in the tergal glands.
In conclusion, my research on the inactive aspartic protease Bla g 2 has led to novel findings including: (i) Bla g 2 mRNA expression profiles in multiple B. germanica tissues, life stages, and sexes, (ii) Bla g 2 protein expression profiles in multiple B. germanica tissues, life stages, and sexes, (iii) Identities of proteins in the tergal gland secretions and tergal glands, (iv) Identities of proteins that co-immunoprecipitate with Bla g 2 in the tergal glands, adult male whole body and gravid female whole body, (v) and annotation of the molecular functions, biological processes, and cellular components proteins found in the tergal glands.