Desmoglein 2 protein, human

Proteomic profiles were characterized using the SOMAscan Assay (SomaLogic, Inc.; Boulder, CO, USA) at the Trans-NIH Center for Human Immunology, Autoimmunity, and Inflammation (CHI), National Institutes of Health (Bethesda, MD, USA). The basis of SOMAscan is built on the use of a new generation of protein-capture Slow Offrate Modified Aptamer (SOMAmer) reagents^{14 (link)}. Using these reagents, the SOMAscan Assay is able to comparatively evaluate protein abundance in 50 μl of serum, plasma, or other biological matrices. Generated by a technique referred to as Selected Evolution of Ligands by Exponential Enrichment (SELEX), the current 1.3 k Assay consists of 1,305 SOMAmer reagents selected against a variety of human proteins (47 % secreted proteins, 28 % extracellular domains, 25 % intracellular proteins) that belong to broad biological subgroups including receptors, kinases, cytokines, proteases, growth factors, protease inhibitors, hormones, and structural proteins.
For serum and plasma samples, SOMAmer reagents are binned into three separate groups according to the expected endogenous abundance of each SOMAmer’s cognate protein in typical human samples. Each SOMAmer reagent exists in only one of the three groupings. Serum and plasma samples (including controls) are then diluted into three concentrations (0.005%, 1%, and 40%) in order to create separate groups for high, medium, and low abundance proteins, respectively. Through this separation, the SOMAscan assay is able to quantify proteins across a dynamic range spanning more than 8 orders of magnitude. The diluted samples are then incubated with the dilution-specific SOMAmers.
Runs in the current 1.3 k Assay were performed semi-automatically with a Tecan Freedom Evo 200 High Throughput System (HTS), which utilizes 96— well plates. In this work, we also present serum runs performed manually with the former 1.1 k Assay using 32— well plates. Supplementary Figure 1(a,b) shows Venn diagram comparisons between the two assays based on aptamer sequence (“SeqId”) and target analyte, respectively. For a total of 1,061 SOMAmers, the aptamer sequence remained unchanged. However, for 60 SOMAmer targets, the aptamer was replaced to improve binding affinity and specificity. The case study presented below in the Results Section presents 1.1 k Assay data from one serum run performed by SomaLogic with a Beckman BioMek Fx HTS. The total number of samples analyzed is 2,624.
The typical SOMAscan plate design includes buffer wells (no sample added), quality control (QC_SOMAscan) and calibrator samples provided by SomaLogic. Quality control and calibrators are pooled samples composed of the same matrix as the biological samples being measured in the plate. Usually, 2 sets of quality control samples are run in duplicate in each plate. Since the intended purpose of these samples is to assess the quality of measurements obtained from one single plate, quality control samples may vary from plate to plate. Also, 5 to 7 replicate calibrator samples are included in each plate with the purpose of normalization across plates. The calibrator consists of a common pooled sample used across a large number of runs; however, when that calibrator lot is depleted, SomaLogic must switch to a different calibrator lot. In addition to these, we have added bridge samples (QC_CHI) to our plate designs in serum and plasma, which we typically run in quadruplicate in each plate and keep consistent across all runs in the 1.3k Assay. Our serum QC_CHI consists of 17 (8 male, 9 female) pooled samples from healthy donors of median age 35 (Q1 = 28.5, Q3 = 54.5) years. For our plasma QC_CHI, we pooled 21 (10 male, 11 female) samples from healthy donors of median age 57 (Q1 = 37, Q3 = 61.5) years. Table 1 presents summary statistics of all serum and plasma runs performed at CHI between January 2015 and April 2017, all of which were analyzed in this paper. The table includes, in parentheses, the breakdown in terms of (n_rep × n_pl), where n_rep is the number of replicates per plate and n_pl is the number of plates.Table 1

Summary statistics of all runs analyzed in this paper.

	Serum 1.3k (HTS)	Plasma 1.3 k (HTS)	Serum 1.1 k (Manual)
Plates	15	8	11
Buffer	19 (1 × 13, 3 × 2)	10 (1 × 7, 3 × 1)	11 (1 × 11)
Calibrators	101 (7 × 13, 5 × 2)	54 (7 × 7, 5 × 1)	55 (5 × 11)
QC_SOMAscan	57 (2 × 27, 3 × 1)	31 (2 × 14, 3 × 1)	22 (2 × 11)
QC_CHI	59 (4 × 14, 3 × 1)	28 (4 × 7)	0

In accordance with SOMAscan’s change log from December 2016, we removed the following 5 SOMAmers throughout: Alkaline phosphatase, tissue-nonspecic isozyme (SeqId 2795-23, UniProt P05186), Complement C1s subcomponent (SeqId 3590-8, UniProt P09871), Desmoglein-2 (SeqId 5071-3, UniProt Q14126), Reticulon-4 (SeqId 5118-74, UniProt Q9NQC3), Tumor necrosis factor receptor super-family member 25 (SeqId 5073-30, UniProt Q93038).

Recombinant human desmoglein-2 protein (DSG2-1601H, Creative Biomart, Shirley, NY) was reconstituted per manufacturer’s instructions and diluted to a concentration of 100 ng/ul with the addition of 1X protease inhibitor (Thermo Fischer Scientific Halt protease inhibitor). The reconstituted protein was then aliquoted and stored at – 80 °C in cryovials for further analysis.
Initial validation of the protein detection was performed using a rabbit monoclonal anti-desmoglein-2 antibodies (DSG2 antibody, Abcam, Waltham, MA) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Abcam, Waltham, MA) as secondary antibodies. Following validation of the recombinant desmoglein-2 protein, optimization of protein detection was performed using various concentrations of primary and secondary antibodies and quantities of recombinant protein. Based on the results of these optimization blots, the protocol for western blot analysis of study samples was developed.
All study samples were analyzed using a standardized protocol in a single laboratory. Recombinant desmoglein-2 proteins (Bio-Rad, Hercules, CA) were first denatured and reduced by boiling in 1 × Laemmli buffer with 2-mercaptoethanol. A total of 375 ng of recombinant desmoglein-2 protein was loaded into each well of precast 4–20% polyacrylamide gels (Mini-protean TGX gels, BioRad, Hercules, CA), alternating with loading of a protein ladder (BioRad, Hercules, CA) and separated by SDS-PAGE before transferring to polyvinylidene difluoride membrane (Immun-blot, BioRad, Hercules, CA) at 60 V for 70 min. Adequate protein transfer was confirmed via Ponceau S solution (Sigma Aldrich, St. Louis, MO). Blots were then blocked with 10% bovine serum albumin (BSA) in Tris buffered saline with 0.05% Tween (TBST) (overnight, 4 °C). Blots were then cropped into sections so that each lane containing the recombinant protein was paired with a protein ladder. Canine sera were thawed at room temperature and diluted 1:100 in TBST with 5% BSA. The cut blots were then incubated in sera from a single study individual for two hours at room temperature, followed by washing in and secondary body incubation with rabbit anti-dog secondary antibody (1:20,000, Abcam, 136759) for one hour at room temperature. After washing, immunoreactive bands were detected using an enhanced chemiluminescence kit (Prometheus ProSignal Dura, Genesee Scientific, El Cajon, CA) and imaged using commercially available imaging system (ProteinSimple FluorChem E system) with an exposure of 60 s. Detected bands were then quantified via densitometry using the ImageJ software correcting for the background density of each blot by a single investigator in a blinded manner.

A Roche LightCycler480 instrument with 384-well microplates was used for this evaluation test of gene expression involved in keratinocytes epidermal physiology. Normal Human Epidermal Keratinocytes were grown and amplified to produce cells for the evaluation. Forty-eight-well microplates were seeded with cells (50,000 cells per well) and incubated for 48 h in a temperature-, humidity-, and CO₂-controlled environment. Samples were added on the cells while renewing the culture medium and were further incubated for 24 h. After incubation, the cells were washed and frozen at −80 °C to preserve the RNA. The RNAs were then extracted and quantified, and their quality was checked before performing their reverse transcription into cDNA. An RT-qPCR was finally performed for each experimental condition for the quantification of the expression of a set of 16 selected genes related to the barrier function (Claudin 1, Cornifelin, Desmoglein 1, Kallikrein-related peptidase 7, Tight junction protein 1), epidermal renewal (Epiregulin, Hyaluronic acid synthase 3, Heparin-binding EGF-like growth factor, Keratin 19), keratinocyte differentiation (Keratin 10, Small prolin-rich protein A1, Transglutaminase 1), and stress response (Glutathione peroxidase 2, Heme oxygenase 1) in keratinocytes. The gene expression was measured on the highest non-toxic dose of each tested sample with a maximal dose of 0.2 g/L or 0.2 mM. The maximum non-cytotoxic dose was determined prior to the gene-expression testing at a dose of 0.2 g/L using a biological model under the same incubation conditions. All samples were evaluated at the same concentration in addition to a five-times-lower dose in a one-step protocol. The fold changes (FC) were calculated after a double normalization against the housekeeping genes and non-treated condition. Fold changes of gene expression were considered as modulated over 1.5 (induction) or under 0.5 (repression).