RESUMO
While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.
Assuntos
Manose/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Progressão da Doença , Glicosilação , Humanos , Espectrometria de Massas em TandemRESUMO
One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.
Assuntos
Glicoproteínas/metabolismo , Calicreínas/metabolismo , Polissacarídeos/metabolismo , Antígeno Prostático Específico/metabolismo , Cromatografia Líquida , Glicosilação , Humanos , Laboratórios , Espectrometria de Massas/métodos , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
In this study we present the design and synthesis of a novel class of peptidomimetics, the ß(3R3)-peptides. Via alternating directions of the amide bonds along ß-peptide sequences, ß(3R3)-peptides can potentially extend the structural space available to ß-peptidic foldamers. Detailed analysis at the air-water interface shows strand conformations and the formation of sheet assemblies with different degrees of crystallinity. Furthermore ß(3R3)-peptides exhibit a high proteolytic stability thus making them an interesting new class of peptidomimetics for biomedical applications.
Assuntos
Peptídeos/síntese química , Peptidomiméticos/síntese química , Água/química , Ar , Conformação Molecular , Peptídeos/química , Peptidomiméticos/química , Estrutura Secundária de ProteínaRESUMO
Pathology differentiation of renal cancer types is challenging due to tissue similarities or overlapping histological features of various tumor (sub) types. As assessment is often manually conducted outcomes can be prone to human error and therefore require high-level expertise and experience. Mass spectrometry can provide detailed histo-molecular information on tissue and is becoming increasingly popular in clinical settings. Spatially resolving technologies such as mass spectrometry imaging and quantitative microproteomics profiling in combination with machine learning approaches provide promising tools for automated tumor classification of clinical tissue sections. In this proof of concept study we used MALDI-MS imaging (MSI) and rapid LC-MS/MS-based microproteomics technologies (15 min/sample) to analyze formalin-fixed paraffin embedded (FFPE) tissue sections and classify renal oncocytoma (RO, n = 11), clear cell renal cell carcinoma (ccRCC, n = 12) and chromophobe renal cell carcinoma (ChRCC, n = 5). Both methods were able to distinguish ccRCC, RO and ChRCC in cross-validation experiments. MSI correctly classified 87% of the patients whereas the rapid LC-MS/MS-based microproteomics approach correctly classified 100% of the patients. This strategy involving MSI and rapid proteome profiling by LC-MS/MS reveals molecular features of tumor sections and enables cancer subtype classification. Mass spectrometry provides a promising complementary approach to current pathological technologies for precise digitized diagnosis of diseases.
RESUMO
The glycome of one of the largest and most exposed human organs, the skin, as well as glycan changes associated with non-melanoma skin cancers have not been studied in detail to date. Skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are among the most frequent types of cancers with rising incidence rates in the aging population. We investigated the healthy human skin N- and O-glycome and its changes associated with BCC and SCC. Matched patient samples were obtained from frozen biopsy and formalin-fixed paraffin-embedded tissue samples for glycomics analyses using two complementary glycomics approaches: porous graphitized carbon nano-liquid chromatography electro spray ionization tandem mass spectrometry and capillary gel electrophoresis with laser induced fluorescence detection. The human skin N-glycome is dominated by complex type N-glycans that exhibit almost similar levels of α2-3 and α2-6 sialylation. Fucose is attached exclusively to the N-glycan core. Core 1 and core 2 type O-glycans carried up to three sialic acid residues. An increase of oligomannose type N-glycans and core 2 type O-glycans was observed in BCC and SCC, while α2-3 sialylation levels were decreased in SCC but not in BCC. Furthermore, glycopeptide analyses provided insights into the glycoprotein candidates possibly associated with the observed N-glycan changes, with glycoproteins associated with binding events being the most frequently identified class.
RESUMO
Production of glycoconjugate vaccines involves the chemical conjugation of glycans to an immunogenic carrier protein such as Cross-Reactive-Material-197 (CRM197). Instead of using glycans from natural sources recent vaccine development has been focusing on the use of synthetically defined minimal epitopes. While the glycan is structurally defined, the attachment sites on the protein are not. Fully characterized conjugates and batch-to-batch comparisons are the key to eventually create completely defined conjugates. A variety of glycoconjugates consisting of CRM197 and synthetic oligosaccharide epitopes was characterised using mass spectrometry techniques. The primary structure was assessed by combining intact protein MALDI-TOF-MS, LC-MALDI-TOF-MS middle-down and LC-ESI-MS bottom-up approaches. The middle-down approach on CNBr cleaved glycopeptides provided almost complete sequence coverage, facilitating rapid batch-to-batch comparisons, resolving glycan loading and identification of side products. Regions close to the N- and C-termini were most efficiently conjugated.
Assuntos
Proteínas de Bactérias/química , Polissacarídeos/metabolismo , Vacinas Conjugadas/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cromatografia Líquida , Epitopos/metabolismo , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vacinas Conjugadas/metabolismoRESUMO
Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two N-glycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab- and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoprotein-receptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcαRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in non-FcαRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo.
Assuntos
Anticorpos Anti-Idiotípicos/uso terapêutico , Receptores ErbB/imunologia , Imunoterapia/métodos , Células Mieloides/metabolismo , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Diferenciação Celular , Receptores ErbB/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos Transgênicos , TransfecçãoRESUMO
Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp ß subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals. BIOLOGICAL SIGNIFICANCE: PCV2-SD is a disease of great economic importance for pig production, characterized by a complex response of the immune system. In the search of a better diagnostic/prognostic marker for porcine PCV2-SD, extensive analyses of the Hp protein backbone and the glycan chains were thoroughly analyzed by various techniques. This resulted in detection and confirmation of Hp O-glycosylation and the glyco-profiling of porcine IgG and IgA. The N- and O-glycosylation of these major porcine plasma glycoproteins appears to be not affected by PCV2-SD infection. Interestingly, these data suggest that this viral infection, which significantly affects the immune responses of the host, leaves the biosynthetic glycosylation processes in the liver and immune cells unaffected. Lack of PTM changes is in contrast to findings in humans where for both proteins pattern changes have been reported in several chronic and inflammatory diseases. This underlines the importance of studying species in detail and not reaching to conclusions by analogy. Furthermore, since Hp is usually quantified by immunoassays in clinical routine analyses, our findings indicate that no bias in Hp determination capabilities due to an altered carbohydrate pattern is to be expected.