Impact of Glycosylation on the Comparability of the Higher-Order Structures in Idursulfase by Hydrogen-Deuterium Exchange Mass Spectrometry.

Characterization of the higher-order structures in idursulfase (iduronate-2-sulfatase, I2S) has been accomplished through the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The method has over 97% sequence coverage, including seven of the eight glycosylation sites, and has been used to study the impact of glycosylation on backbone proton exchange. In addition, the method adapted a well-used biophysical spectra comparison method (similarity scoring) to define quantitative acceptance criteria for analytical comparability of different batches of drug substance as well as samples with modulated glycans. Differences in the HDX profile were induced by enzymatic removal of terminal sialic and phosphate groups on negatively charged glycans. These differences were mapped to the crystal structure and demonstrated synergistic HDX changes focused around the N221 and N255 glycosylation sites, which contain mannose-6-phosphate motifs important for I2S uptake into cells.

Functional role and therapeutic targeting of p21-activated kinase 4 in multiple myeloma.

Dysregulated oncogenic serine/threonine kinases play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4) and its potential as a new target in MM for clinical applications. PAK4 promoted MM cell growth and survival via activation of MM survival signaling pathways, including the MEK-extracellular signal-regulated kinase pathway. Furthermore, treatment with orally bioavailable PAK4 allosteric modulator (KPT-9274) significantly impacted MM cell growth and survival in a large panel of MM cell lines and primary MM cells alone and in the presence of bone marrow microenvironment. Intriguingly, we have identified FGFR3 as a novel binding partner of PAK4 and observed significant activity of KPT-9274 against t(4;14)-positive MM cells. This set of data supports PAK4 as an oncogene in myeloma and provide the rationale for the clinical evaluation of PAK4 modulator in myeloma.

The application of HPLC/MS analysis with a multi-enzyme digest strategy to characterize different interferon product variants produced from Pichia pastoris.

Interferons are signaling proteins that belong to the large class of cytokines and human interferons which are classified based on the type of receptor interactions: type I, II and III. IFNα2b belongs to the type I interferon class with a major therapeutic application for the treatment of hepatitis B and C infections. A recombinant form of IFNα2b expressed in E. coli, known as IntronA, has been approved by US Food and Drug Administration (FDA). IFN γ, also known as type II interferon, plays a significant role in the inhibition of viral replication. Actimmune® is a US Food and Drug Administration (FDA) approved version of IFN γ for the indication of reducing infections associated with chronic granulomatous disease and severe malignant osteopetrosis. In this study we have applied advanced analytical methods for the characterization of IFNα2b and IFN γ produced from Pichia pastoris. The multi-enzyme digestion approach has been developed to allow measurement of 100% sequence coverage and detailed analysis of post-translational variants and degradation products. In this manner, we identified the following variants in IFN α2b: N-terminal residual leader sequence, an amino acid substitution, oxidation of methionine residues and two sites of high mannose N-glycosylation. In the Pichia IFN γ produced material, our approach detected variants resulting from glycosylation, C-terminal proteolysis, oxidation of methionine residues and deamidation. In this manner, the analytical program was able to support rapid process development as well as identify product variants and degradation products in the resulting product.

Characterization of Site-Specific Glycosylation in Influenza A Virus Hemagglutinin Produced by Spodoptera frugiperda Insect Cell Line.

Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion was used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN, and pepsin, were used separately to generate suitable glycopeptides for online LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation, and the peptide backbone information was provided by collision-induced dissociation (CID)-MS3 fragmentation. With this approach, 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed, and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.

Integrated Bottom-Up and Top-Down Liquid Chromatography-Mass Spectrometry for Characterization of Recombinant Human Growth Hormone Degradation Products.

With the advent of biosimilars to the U.S. market, it is important to have better analytical tools to ensure product quality from batch to batch. In addition, the recent popularity of using a continuous process for production of biopharmaceuticals, the traditional bottom-up method, alone for product characterization and quality analysis is no longer sufficient. Bottom-up method requires large amounts of material for analysis and is labor-intensive and time-consuming. Additionally, in this analysis, digestion of the protein with enzymes such as trypsin could induce artifacts and modifications which would increase the complexity of the analysis. On the other hand, a top-down method requires a minimum amount of sample and allows for analysis of the intact protein mass and sequence generated from fragmentation within the instrument. However, fragmentation usually occurs at the N-terminal and C-terminal ends of the protein with less internal fragmentation. Herein, we combine the use of the complementary techniques, a top-down and bottom-up method, for the characterization of human growth hormone degradation products. Notably, our approach required small amounts of sample, which is a requirement due to the sample constraints of small scale manufacturing. Using this approach, we were able to characterize various protein variants, including post-translational modifications such as oxidation and deamidation, residual leader sequence, and proteolytic cleavage. Thus, we were able to highlight the complementarity of top-down and bottom-up approaches, which achieved the characterization of a wide range of product variants in samples of human growth hormone secreted from Pichia pastoris.

Comparative study of profiling post-translational modifications of a circulating antibody drug in human with different capture reagents.

Capture reagents are critical to affinity-based bioanalytical methods. The potential bias of capture reagents, for or against certain subpopulations of the target of interest, may lead to inaccurate quantitation. This issue is more profound for sensitive measurements, such as post-translational modification (PTM) profiling of therapeutic proteins from complex matrix. Here, a recently developed affinity purification coupled mass spectrometric method was utilized to assess the full sequence of a circulating therapeutic aglycosylated IgG1 (MAB3) in human subject, using two different capture reagents. We monitored all PTMs known to be related to MAB3 drug quality (three representative PTMs are shown in this paper). The results validated the comparability of these two reagents.

Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation.

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.

Identification of Low Abundant Isomeric N-Glycan Structures in Biological Therapeutics by LC/MS.

An effective LC-MS based method for online characterization of low abundant structural isomers of N-linked glycans in biological therapeutics was developed. N-linked glycans of a recombinant monoclonal antibody were released by PNGase F and labeled with 2-aminobenzamide (2-AB) fluorescent tag. The labeled glycans were analyzed by online ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC) coupled with mass spectrometry (MS). The glycan structure was characterized by MS(n) fragmentation in negative ion mode followed by identification of the signature D ions. The assignment included monosaccharide sequence and linkage information. The developed method successfully characterized structural isomers of A1G1F (assigned as terminal sialic acid attached in the 1,6 branch at 2,3 position), and A1G1F' (assigned as terminal sialic acid attached in the 1,3 branch at 2,3 position). Moreover, using the same approach, previously unknown low abundant species were identified unambiguously. One such structural isomer at low level, terminal GlcNAc of G1F+GlcNAc, was identified to be linked at the 1,6 branch. Additionally, another low level structural isomer, previously assigned as Man8 glycan, was found to be Man7+Glc glycan as its 1,3 branch containing three mannoses and one terminal glucose. The identification was further confirmed by a purified α-1,2-endomannosidase enzyme to generate the cleavage of α-1,3 linked terminal disaccharides (Man+glucose). Using this approach, different lots or different CHO produced mAbs was thoroughly examined and found that the newly identified "Man8" (Man7+Glc) was also present in different batches and in some commercially available therapeutic mAbs.

Aberrant serum immunoglobulin G glycosylation in chronic hepatitis B is associated with histological liver damage and reversible by antiviral therapy.

BACKGROUND: Aberrant serum immunoglobulin G (IgG) glycosylation and its immunomodulatory effect are rarely addressed in chronic hepatitis B virus (HBV) infection. METHODS: Serum IgG-Fc glycosylation profiles in 76 patients with HBV-related liver cirrhosis and 115 patients with chronic hepatitis B (CHB) before and after 48 weeks of anti-HBV nucleos(t)ide analogue treatment were analyzed using high-throughput liquid chromatography-mass spectrometry and were compared to profiles in 108 healthy controls. RESULTS: The level of aberrant serum IgG-Fc glycosylation ,: particularly galactose deficiency, was higher in patients with CHB and those with cirrhosis (P < .001 for both) than in healthy controls. IgG galactose deficiency was correlated with the severity of liver necroinflammation and fibrosis in CHB. Multivariate logistic regression analyses showed that the IgG-Fc glycoform with fucosylation and fully galactosylation was an independent factor for a total Knodell necroinflammation score of ≥ 7 (odds ratio, 0.74; 95% confidence interval, .56-.97) and an Ishak fibrosis score of ≥ 3 (odds ratio, 0.69; 95% confidence interval, .49-.97). Administration of antiviral therapy for 48 weeks reversed aberrant IgG-Fc glycosylation in patients with CHB from week 12 onward but did not reverse glycosylation in patients with cirrhosis. Attenuated IgG opsonization in patients with CHB, which was correlated with aberrant Fc-glycosylation, was reversed after treatment as well. CONCLUSIONS: Aberrant serum IgG-Fc glycosylation in CHB, which is highly associated with histological liver damage, affects IgG opsonizing activity and can be reversed by antiviral therapy.

Integrated Proteomic and Genomic Analysis of Gastric Cancer Patient Tissues.

V-erb-b2 erythroblastic leukemia viral oncogene homologue 2, known as ERBB2, is an important oncogene in the development of certain cancers. It can form a heterodimer with other epidermal growth factor receptor family members and activate kinase-mediated downstream signaling pathways. ERBB2 gene is located on chromosome 17 and is amplified in a subset of cancers, such as breast, gastric, and colon cancer. Of particular interest to the Chromosome-Centric Human Proteome Project (C-HPP) initiative is the amplification mechanism that typically results in overexpression of a set of genes adjacent to ERBB2, which provides evidence of a linkage between gene location and expression. In this report we studied patient samples from ERBB2-positive together with adjacent control nontumor tissues. In addition, non-ERBB2-expressing patient samples were selected as comparison to study the effect of expression of this oncogene. We detected 196 proteins in ERBB2-positive patient tumor samples that had minimal overlap (29 proteins) with the non-ERBB2 tumor samples. Interaction and pathway analysis identified extracellular signal regulated kinase (ERK) cascade and actin polymerization and actinmyosin assembly contraction as pathways of importance in ERBB2+ and ERBB2- gastric cancer samples, respectively. The raw data files are deposited at ProteomeXchange (identifier: PXD002674) as well as GPMDB.

Constitutively oxidized CXXC motifs within the CD3 heterodimeric ectodomains of the T cell receptor complex enforce the conformation of juxtaposed segments.

The CD3ϵγ and CD3ϵδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αß heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ϵ, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction.

Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.

Identification of novel glycans with disialylated structures in α3 integrin from mouse kidney cells with the phenotype of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder caused by mutations in the Pkd1 or Pkd2 genes, in which large cysts replace normal kidney tissue, leading to end-stage kidney disease. In this study we have utilized a powerful nano-HPLC-mass spectrometric approach to characterize patterns of normal and abnormal N-linked glycosylation of α3 integrin subunit in Pkd1(-/-) cells derived from mouse kidneys. Higher molecular weight glycan structures with a different monosaccharide composition were observed at two sites, namely, Asn-925 and Asn-928 sites in α3 integrin isolated from Pkd1(+/+) cells compared with Pkd1(-/-) cells. In addition, an unusual and unique disialic acid glycan structure was observed solely in Pkd1(-/-) cells. Thus, these studies suggest that abnormal protein glycosylation may have a role on the pathogenesis of cyst formation in ADPKD.

Distinct splice variants and pathway enrichment in the cell-line models of aggressive human breast cancer subtypes.

This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes; it contains many cancer-associated genes, including BRCA1, ERBB2, (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell-line models of hormone-receptor-negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (overexpression)/ER-/PR-; adenocarcinoma), SUM190 (ERBB2+ (overexpression)/ER-/PR-; inflammatory breast cancer), and SUM149 (ERBB2 (low expression) ER-/PR-; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared with the other two cancer cell lines and with normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell-to-cell adhesion, integrin, and ERK1/ERK2 signaling; and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 overexpressed cell line models and an association of nucleotide binding, RNA splicing, and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct variants identified from each of these three breast cancer cell line models that may open opportunities for drug target discovery and help unveil their specific roles in cancer progression and metastasis.

Genome wide proteomics of ERBB2 and EGFR and other oncogenic pathways in inflammatory breast cancer.

In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with reads per kilobase per million mapped reads (RPKM) values for ERBB2 (14.4, 400, and 300 for SUM149, SUM190, and SKBR3, respectively) and for EGFR (60.1, not detected, and 1.4 for the same 3 cell lines). We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g., 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used bioinformatics sites GeneGo, PathwayCommons and NCI receptor signaling networks to identify pathways that contained the four main oncogenes and had good coverage in the transcriptomic and proteomic data sets as well as a significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling; caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings; branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 subpathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.

A chromosome-centric human proteome project (C-HPP) to characterize the sets of proteins encoded in chromosome 17.

We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer-associated genes, we have focused the clustering of cancer-associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of coexpression through coordinated regulation.

Structure of Sad1-UNC84 homology (SUN) domain defines features of molecular bridge in nuclear envelope.

The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.

LC-MS analysis of polyclonal human anti-Neu5Gc xeno-autoantibodies immunoglobulin G Subclass and partial sequence using multistep intravenous immunoglobulin affinity purification and multienzymatic digestion.

Human polyclonal IgG antibodies directly against the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) are potential biomarkers and mechanistic contributors to cancer and other diseases associated with chronic inflammation. Using a sialoglycan microarray, we screened the binding pattern of such antibodies (anti-Neu5Gc IgG) in several samples of clinically approved human IVIG (IgG). These results were used to select an appropriate sample for a multistep affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multienzyme digestion procedure followed by nano liquid chromatography (nanoLC) coupled to linear ion trap-Fourier transform mass spectrometry (LTQ-FTMS). We used characteristic and unique peptide sequences to determine the IgG subclass distribution and thus provided direct evidence that all four IgG subclasses can be generated during a xeno-autoantibody immune response to carbohydrate Neu5Gc-antigens. Furthermore, we obtained a significant amount of sequence coverage of both the constant and variable regions. The approach described here, therefore, provides a way to characterize these clinically significant antibodies, helping to understand their origins and significance.

Reversion of the ErbB malignant phenotype and the DNA damage response.

The ErbB or HER family is a group of membrane bound tyrosine kinase receptors that initiate signal transduction cascades, which are critical to a wide range of biological processes. When over-expressed or mutated, members of this kinase family form homomeric or heteromeric kinase assemblies that are involved in certain human malignancies. Targeted therapy evolved from studies showing that monoclonal antibodies to the ectodomain of ErbB2/neu would reverse the malignant phenotype. Unfortunately, tumors develop resistance to targeted therapies even when coupled with genotoxic insults such as radiation. Radiation treatment predominantly induces double strand DNA breaks, which, if not repaired, are potentially lethal to the cell. Some tumors are resistant to radiation treatment because they effectively repair double strand breaks. We and others have shown that even in the presence of ionizing radiation, active ErbB kinase signaling apparently enhances the repair process, such that transformed cells resist genotoxic signal induced cell death. We review here the current understanding of ErbB signaling and DNA double strand break repair. Some studies have identified a mechanism by which DNA damage is coordinated to assemblies of proteins that associate with SUN domain containing proteins. These assemblies represent a new target for therapy of resistant tumor cells.

Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation.

The disulfides in three monoclonal antibodies (mAb), the anti-HER2, anti-CD11a, and GLP-1 with IgG4-Fc fusion protein, were completely mapped by LC-MS with the combination of electron-transfer dissociation (ETD) and collision induced dissociation (CID) fragmentation. In addition to mapping the 4 inter- and 12 intrachain disulfides (total 16), the identification of scrambled disulfides in degraded samples (heat-stress) was achieved. The scrambling was likely attributed to an initial breakage between the light (Cys 214) and heavy (Cys 223) chains in anti-HER2, with the same observation found in a similar therapeutic mAb, anti-CD11a. On the other hand, the fusion antibody, with no light chain but containing only two heavy chains, generated much less scrambling under the same heat-stressed conditions. The preferred sites of scrambling were identified, such as the intrachain disulfide for CxxC in the heavy chain, and the C194 of the heavy chain pairing with the terminal Cys residue (C214) in the light chain. The interchain disulfides between the light and heavy chains were weaker than the interchain disulfides between the two heavy chains. The relative high abundance ions observed in ETD provided strong evidence for the linked peptide information, which was particularly useful for the identification of the scrambled disulfides. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) helped the separation of these misfolded proteins for the determination of scrambled disulfide linkages. This methodology is useful for comparison of disulfide stability generated from different structural designs and providing a new way to determine the scrambling patterns, which could be applied for those seeking to determine unknown disulfide linkages.