Sulfated Glycoprotein Research Articles

Microanatomy of the Testes and Male Genital Ducts of Banded Krait Bungarus fasciatus (Schneider, 1801)

The Banded krait is a species of venomous snakes that ranks among the top 5 in Thailand, with a significance role as a predator for controlling other animals. Despite numerous studies on the ecology, the microanatomy of body systems has still not reported clearly. The present study was to investigate the microanatomy of the testes and male genital ducts of Banded krait that collected from Phatthalung Province, Thailand. The snakes were collected after anesthetized, and their testes and genital ducts were fixed in Bouin’s fixative. The reproductive organs were collected during July to October 2022, and were sectioned of 5-µm-thick pieces for staining with Harris’s hematoxylin and eosin (H&E), periodic acid Schiff’s (PAS), alcian blue (AB) pH 2.5 and pH 1.0, bromophenol blue (BB) and Masson’s trichrome (MT). The testes contained the seminiferous tubules with 7 stages of germ cells: (I) type A spermatogonium, (II) type B spermatogonium, (III) primary spermatocyte, (IV) secondary spermatocyte, (V) round spermatid, (VI) elongated spermatid and (VII) spermatozoa. Besides, Sertoli and Leydig’s cells were appeared in the seminiferous tubules and the interstitial area, respectively. During the sampling period, the spermatozoa were observed in the seminiferous tubules and genital ducts. The extra-testicular rete testis, ductuli efferentes and ductus epididymis were embedded in the epididymal sheath. The epithelial cells of rete testis, ductus epididymis, as well as ductus deferens possessed microvilli, whereas those of the ductuli efferentes composed of cilia. The epithelial cells of all portions showed the positive staining with PAS and BB due to the presence of neutral glycoproteins and proteins, respectively, and the negative staining for AB pH 2.5 and pH 1.0 because of the absence of acidic glycoproteins. HIGHLIGHTS During the sampling period from July to October, the Banded Krait exhibited active spermatogenesis The rete testis could be divided into 2 portions: intra-rete testis and extra-rete testis The ductus epididymis was divided into 3 portions: proximal, middle and distal ductus epididymis All portions of the male genital ducts, the epithelium showed the secretion of neutral glycoproteins and proteins without sulfated and carboxylated glycoproteins GRAPHICAL ABSTRACT

The role of Clec11a in bone construction and remodeling.

Bone is a dynamically active tissue whose health status is closely related to its construction and remodeling, and imbalances in bone homeostasis lead to a wide range of bone diseases. The sulfated glycoprotein C-type lectin structural domain family 11 member A (Clec11a) is a key factor in bone mass regulation that significantly promotes the osteogenic differentiation of bone marrow mesenchymal stem cells and osteoblasts and stimulates chondrocyte proliferation, thereby promoting longitudinal bone growth. More importantly, Clec11a has high therapeutic potential for treating various bone diseases and can enhance the therapeutic effects of the parathyroid hormone against osteoporosis. Clec11a is also involved in the stress/adaptive response of bone to exercise via mechanical stimulation of the cation channel Pieoz1. Clec11a plays an important role in promoting bone health and preventing bone disease and may represent a new target and novel drug for bone disease treatment. Therefore, this review aims to explore the role and possible mechanisms of Clec11a in the skeletal system, evaluate its value as a potential therapeutic target against bone diseases, and provide new ideas and strategies for basic research on Clec11a and preventing and treating bone disease.

Cathartocytosis: How Cells Jettison Unwanted Material as They Reprogram.

Injury can cause differentiated cells to undergo massive reprogramming to become proliferative to repair tissue via a cellular program called paligenosis. Gastric digestive-enzyme-secreting chief cells use paligenosis to reprogram into progenitor-like Spasmolytic-Polypeptide Expressing Metaplasia (SPEM) cells. Stage 1 of paligenosis is to downscale mature cell architecture via a process involving lysosomes. Here, we noticed that sulfated glycoproteins (which are metaplasia and cancer markers in mice and humans) were not digested during paligenosis but excreted into the gland lumen. Various genetic and pharmacological approaches showed that endoplasmic reticulum membranes and secretory granule cargo were also excreted and that the process proceeded in parallel with, but was independent lysosomal activity. 3-dimensional light and electron-microscopy demonstrated that excretion occurred via unique, complex, multi-chambered invaginations of the apical plasma membrane. As this lysosome-independent cell cleansing process does not seem to have been priorly described, we termed it "cathartocytosis". Cathartocytosis allows a cell to rapidly eject excess material (likely in times of extreme stress such as are induced by paligenosis) without waiting for autophagic and lysosomal digestion. We speculate the ejection of sulfated glycoproteins (likely mucins) would aid in downscaling and might also help bind and flush pathogens (like H pylori which causes SPEM) away from tissue.

Abstract 4122: Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis

Abstract INTRODUCTION: Metastasis is the major cause of cancer-related deaths (90%). Intercellular communication between cancer cells and with cells in their environment plays a significant function in tumor progression. In fact, secreted factors like cytokines and extracellular vesicles (EVs) can either halt or stimulate tumorigenesis as well as metastasis. We have previously reported, that the integrin B3 (ITGB3) is required for metastasis of breast cancer cells in vivo (Sese et al. Oncotarget, 2017). We furthermore demonstrated, that EVs mediated colony growth is dependent on ITGB3 and that endocytosis mediated uptake of EVs relies on the interaction of ITGB3 with integrin av (ITGav) and heparan sulphated glycoproteins (HSPGs) (Fuentes et al. Nat. Commun., 2021). OBJECTIVE: The objective of this work is focused on identifying inhibitors targeting the ITGav/B3/HSPGs complex to prevent extracellular vesicle uptake in cancer cells and to test their ability to prevent metastasis. METHODOLOGY. Inhibitors targeting ITGB3/ITGAV or HSPGs were either commercially available or obtained through collaborations and single chain antibodies (scFv) were produced in house. Inhibition of fluorescent labeled EVs was determined by FACS analysis or Incucyte based measurements and the efficacy of inhibitors was determined in a dose dependent manner. Toxicity of inhibitors was studied by crystal violet-based assay in normal and tumor cell lines. Tail vein injection of luciferase labeled MDA-MB-231 cells into nude mice was used as model to study metastatic diseases under treatment with the identified inhibitors. Metastasis formation was monitored by bioluminescence imaging and lungs were examined pathologically at end point. Overall survival was determined. RESULTS: Among the tested Integrin and HSPGs targeting compounds, we identified an integrin-binding antibody, an integrin binding scFv, an RGD-mimetic compound and an HSPGs-binding peptide as inhibitors of EV uptake. Inhibition was validated in a dose dependent manner and toxicity studies did not show adverse effects. Three of those inhibitors, targeting either ITGB3/ITGAV or HSPGs were tested in mice models in vivo and all inhibitors could significantly reduce the formation of metastasis and strongly increase the overall survival. CONCLUSION: We have identified four drug candidates that target the ITGav/B3/HSPGs complex and are able to prevent EV uptake, demonstrating that EV uptake is a druggable process. Even more, the inhibitors tested in vivo could strongly reduce metastasis formation and increase overall survival. This data supports our hypothesis, that targeting EV mediated intercellular communication is a valuable approach to combat cancer metastasis. Citation Format: Elena Muro-Blanc, Rocío Bayona-Ramón y Cajal, Marta Cano-Galietero, Sara García-Ortega, Chiara Falciani, Luisa Bracci, Alberto J. Schuhmacher, Stefan Hümmer, Santiago Ramón y Cajal. Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4122.

Biochemical characterization, and anti-inflammatory and antitumor activities of glycoprotein from lamb abomasum.

Lamb abomasum is used as an edible medicinal source in traditional Chinese medicine for the treatment of gastrointestinal disorders. Lamb abomasum sourced biochemical drug Lamb's trip extract and Vitamin B12 capsule used for the clinical treatment of chronic gastritis, gastric ulcer, and reversal of intestinal metaplasia. Therefore, claimed to have prevention of gastric cancer activity. In this study, we aim to assess whether the glycoprotein has biological activity in the cure of gastric disorder and conduct a structure-activity relationship. Glycoproteins' extraction conditions were optimized by the response surface method and purified with DEAE-cellulose and Sephadex G-50 chromatography. Two homogenous glycoproteins' physiochemical structures were studied with electrophoresis, HPLC analysis, peroxide oxidation, and β-elimination, FT-IR, CD, LC-MS/MS, and EDS analysis. The antiinflammation activity of the glycoprotein was determined against COX-2 and LOX-15 enzyme inhibitory ability in vitro, and antitumor activity against HT-29 and HGC-25, and cytotoxicity on L-02cells was determined in vivo with the MTT method. The abomasum was abundant in glycoprotein and the extraction yield of glycoprotein was up to 24.6±2.1% under optimized conditions. Two homogeneous glycoproteins SAGP-I and SAGP-II determined to be ribose-conjugated and sulfated glycoproteins with a molecular weight of 15.6kDa and 6.4kDa. And according to the structural analysis, SAGP-I was a mucin-type ribose-conjugated glycoprotein with 14 O-glycosylation and one N- glycosylation site. SAGP-I and SAGP-II have remarkable anti-inflammatory activity against COX-2 enzyme with the IC50 of 17.64±1.25μg/mL and 16.14±1.11μg/mL, respectively. Meanwhile, the two glycoproteins showed strong antitumor activity against HT-29 with the EC50 of 19.19±1.46μg/mL and 184.9±5.6μg/mL, respectively. The Highly purified glycoprotein SAGP-1 and SAGP-II showed anti-inflammatory activity against the COX-2 enzyme, and antitumor activity against HT-29 human colon cancer cells and noun-inhibitory activity against LOX-15 enzyme and HGC-25. Both glycoproteins are ribose conjugated and sulfated whose characters are related to their anti-inflammatory and anti-tumor activity. Such results suggest the possibility of anti-inflammatory and pre-cancer activity. And in some degree explains the pharmacy of abomasum's traditional use in gastric disorder and clinical use of lamb abomasum APIs drugs' in gastric disorders and gastric cancer development. This study provides a preliminary basis for the further study of the per-cancer mechanism of lamb abomasum glycoprotein. And, would be the material basis of the clinical use of Lamb's trip extract and Vitamin B12 capsule.

Clusterin and Its Isoforms in Oral Squamous Cell Carcinoma and Their Potential as Biomarkers: A Comprehensive Review.

Oral squamous cell carcinoma (OSCC) is a prevalent type of head and neck cancer, ranked as the sixth most common cancer worldwide, accounting for approximately 300,000 new cases and 145,000 deaths annually. Early detection using biomarkers significantly increases the 5-year survival rate of OSCC by up to 80-90%. Clusterin (CLU), also known as apolipoprotein J, is a sulfated chaperonic glycoprotein expressed in all tissues and human fluids and has been reported to be a potential biomarker of OSCC. CLU has been implicated as playing a vital role in many biological processes such as apoptosis, cell cycle, etc. Abnormal CLU expression has been linked with the development and progression of cancers. Despite the fact that there are many studies that have reported the involvement of CLU and its isoforms in OSCC, the exact roles of CLU and its isoforms in OSCC carcinogenesis have not been fully explored. This article aims to provide a comprehensive review of the current understanding of CLU structure and genetics and its correlation with OSCC tumorigenesis to better understand potential diagnostic and prognostic biomarker development. The relationship between CLU and chemotherapy resistance in cancer will also be discussed to explore the therapeutic application of CLU and its isoforms in OSCC.

Hallmarks of the relationship between host and Trypanosoma cruzi sulfated glycoconjugates along the course of Chagas disease.

American Trypanosomiasis or Chagas disease (ChD), a major problem that is still endemic in large areas of Latin America, is caused by Trypanosoma cruzi. This agent holds a major antigen, cruzipain (Cz). Its C-terminal domain (C-T) is retained in the glycoprotein mature form and bears several post-translational modifications. Glycoproteins containing sulfated N-linked oligosaccharides have been mostly implicated in numerous specific procedures of molecular recognition. The presence of sulfated oligosaccharides was demonstrated in Cz, also in a minor abundant antigen with serine-carboxypeptidase (SCP) activity, as well as in parasite sulfatides. Sulfate-bearing glycoproteins in Trypanosomatids are targets of specific immune responses. T. cruzi chronically infected subjects mount specific humoral immune responses to sulfated Cz. Unexpectedly, in the absence of infection, mice immunized with C-T, but not with sulfate-depleted C-T, showed ultrastructural heart anomalous pathological effects. Moreover, the synthetic anionic sugar conjugate GlcNAc6SO3-BSA showed to mimic the N-glycan-linked sulfated epitope (sulfotope) humoral responses that natural Cz elicits. Furthermore, it has been reported that sulfotopes participate via the binding of sialic acid Ig-like-specific lectins (Siglecs) to sulfosialylated glycoproteins in the immunomodulation by host-parasite interaction as well as in the parasite infection process. Strikingly, recent evidence involved Cz-sulfotope-specific antibodies in the immunopathogenesis and infection processes during the experimental ChD. Remarkably, sera from chronically T. cruzi-infected individuals with mild disease displayed higher levels of IgG2 antibodies specific for sulfated glycoproteins and sulfatides than those with more severe forms of the disease, evidencing that T. cruzi sulfotopes are antigenic independently of the sulfated glycoconjugate type. Ongoing assays indicate that antibodies specific for sulfotopes might be considered biomarkers of human cardiac ChD progression, playing a role as predictors of stability from the early mild stages of chronic ChD.

Recognition of Tumor Nidogen-1 by Neutrophil C-Type Lectin Receptors.

Neutrophil-mediated cytotoxicity toward tumor cells requires cell contact and is mediated by hydrogen peroxide. We have recently shown that Cathepsin G expressed on the neutrophil surface interacts with tumor RAGE, and this interaction facilitates neutrophil cytotoxicity. Interruption of the Cathepsin G–RAGE interaction led to 50–80% reduction in cytotoxicity, suggesting that additional interactions are also involved. Here we show that blocking antibodies to the C-type lectin receptors (CLRs) Clec4e and Dectin-1, but not those to NKG2D, attenuated murine neutrophil cytotoxicity towards murine tumor cells, suggesting a contributing role for these CLRs in neutrophil recognition of tumor cells. We further observed that the CLRs interact with tumor Nidogen-1 and Hspg2, two sulfated glycoproteins of the basement membrane. Both Nidogen-1 and Hspg2 were found to be expressed on the tumor cell surface. The knockdown of Nidogen-1, but not that of Hspg2, led to reduced susceptibility of the tumor cells to neutrophil cytotoxicity. Altogether, this study suggests a role for CLR–Nidogen-1 interaction in the recognition of tumor cells by neutrophils, and this interaction facilitates neutrophil-mediated killing of the tumor cells.

A Novel Sulfated Glycoprotein Elicitor Extracted from the Moroccan Green Seaweed Codium decorticatum Induces Natural Defenses in Tomato

Sulfated glycoproteins extracted for the first time from the Moroccan green seaweed Codium decorticatum were investigated for their ability to induce a natural defense metabolism in the roots and the upper leaves of tomato seedlings. The crude (AGB) and the purified fractions (AGP) were characterized chemically (Colorimetric assays) and structurally (SEC-MALS, GC-EI/MS, ATR-FTIR). The elicitor aqueous solutions (1 g/L) were applied by foliar spray and syringe infiltration into the internodal middle of 45-day-old tomato seedlings. Phenylalanine ammonia-lyase (PAL) activity, polyphenols, and lignin contents were measured in the roots and the leaves after 0 h, 12, 24, 48, and 72 h of treatment. The AGB and AGP extracts contained 37.67% and 48.38% of the total carbohydrates, respectively, and were mainly composed of galactose, glucose, arabinose, and a minor amount of xylose and rhamnose. They were characterized by an important molecular weight (Mw) > of 2000 × 103 g·mol−1 and a high degree of sulfation and protein (12–23% (w/w)), indicating that the extracted polysaccharides could be an arabinogalactan-rich protein present in the cell wall of the green seaweed C. decorticatum. Both crude and purified fractions exhibited an elicitor effect by inducing the PAL activity, the accumulation of phenolic compounds and lignin contents in the roots and the leaves of tomato seedlings. These responses were systemic in both the methods used (injection and foliar spray) and were mobilized throughout tissues that are not directly treated (roots and/or leaves). Regarding the elicitor activities, AGB and AGP presented globally similar patterns, which revealed the importance of crude extracts in the stimulation of plant immunity. These results suggest the new application of sulfated glycoprotein isolated from green seaweed in agriculture as inducers of natural defenses of plants.

Sulfated Glycoprotein Synthesis.

Chemical protein synthesis has achieved tremendous progress in the past decades. With the development of chemical ligation as powerful tools, the scope of synthetic protein is greatly expanded. Proteoglycans are a class of sulfated glycoproteins widely distributed on the cell surface and in the extracellular matrix, which are extensively engaged in cellular communication events. Consisting of protein backbone and glycosaminoglycan(s) side chain, proteoglycans are highly complex and heterogeneous in nature. Chemical synthesis provides facile and reliable approach to these molecules, with defined glycan structure and sulfation pattern. One remaining problem is that the acid-labile sulfates could hardly survive during the typical solid phase peptide synthesis (SPPS) process. In this chapter, strategic design of a "glycopeptide cassette" for the preparation of sulfated glycoprotein is described. In particular, we provide protocols for the chemical synthesis of ectodomain fragment (23-120) of sulfated glycoprotein syndecan-1.

Pathobiochemical pathways of redox imbalance in the neurological long-term effects of COVID-19 and the role of chondroitin sulfate in the redox status restoration

The review examines the epidemiology and clinical manifestations of COVID-19 long-term neurological effects, main pathobiochemical mechanisms, and integrated circuits of redox status impairment in COVID-19, such as the decrease of adenosine triphosphate production, fatty acids levels, acylcarnitine, and amino acids, impairment of oxidative phosphorylation and glycolysis, hypometabolic state, redox imbalance with the increase of peroxides and superoxides, isoprostanes, the decrease of α-tocopherol, substances reacting with thiobarbituric acid, increased nitrosative stress with the increase of inducible synthase of nitric oxide, nitric oxide, peroxynitrite, and nitrate. Neuroprotective approaches aimed at suppressing excitotoxicity, oxidative stress, and neuroinflammation are presented. Recent data on the relationship between mechanisms of chondroitin sulfate and its derivatives (chondroitin sulfate glycoprotein disaccharide) neuroprotective effects and characteristics of their chemical structure are analyzed. The mechanism of action and neuroprotective effects of chondroitin sulfate and its derivatives in fatigue syndrome in patients with SARS-CoV2 infection are discussed (regulation of the PKC/PI3K/Akt activity, the increase of heme oxygenase-1 level, the decrease of reactive oxygen species). The position that chondroitin sulfate and its derivatives can become promising drugs to prevent the long-term neurological effects of COVID-19 is reasoned.

Deregulated molecular genetic pathways in urinary bladder cancer with the importance of molecular biomarkers in diagnosis and follow-up- A comprehensive and updated review

This article reviews and compares various currently available non-invasive, molecular biomarker-based tests for bladder cancer (BC) detection, and evaluates other potential molecules that may be used for BC diagnosis and surveillance. Currently, urinary cytology and periodic cystoscopies are clinically recommended for the diagnosis and monitoring of BC. Though highly specific, urinary cytology lacks sensitivity, whereas cystoscopies are invasive tests that are uncomfortable for the patient. Molecular biomarkers associated with BC progression form the basis for various available non-invasive tests. Urinary proteins like NMP22, MCM5 and Human complement factor-H related protein (BTA) are detected by ELISA/Immunochromatographic assay devices (NMP22, BTA, ADXBLADDER). Chromosomal abnormalities reported in BC -aneuploidy in chromosomes 3, 7, and 17 and deletions in chromosome 9- are detected by the Fluorescence in situ Hybridization based UroVysion test. Tumor antigens like sulfated mucin glycoproteins and glycosylated CEA help to light up urothelial carcinoma cells exfoliated into urine (uCyt+). Mutations in TERTp, FGFR3, KRAS, HRAS, TP53, CDKN2A, ERBB2 and PIK3CA contribute to BC progression and these are detected in tumor DNA by RT-qPCR, NGS and/or Sanger sequencing-based assays (Uromonitor, UroSEEK, AssureMDX). mRNA levels of genomic markers frequently deregulated in BC like IGFBP5, HOXA13, MDK, CDC2, CXCR5, IGF2, ABL1, CRH, UPK1B and ANXA10 are assesed by CxBladder and Xpert Bladder Cancer Monitor. BC associated changes in DNA methylation are detected by real time PCR and NGS based assays (EpiCheck, UroMark, AssureMDX). These tests have not yet been formally indicted into clinical practice but can serve as sensitive indicators for early diagnosis, disease monitoring, and treatment response in BC. Keywords: bladder cancer, biomarkers, non-invasive tests, molecular events in bladder cancer, genetic changes in bladder cancer

Glycoprotein Aging with Increased Mannose Exposure Linked to Cardiovascular Disease through the Macrophage Mannose Receptor (Mrc1)

N‐glycosylation is common to the vast majority of secretory proteins that are found at the cell surface or secreted. We have previously discovered that as secreted glycoproteins age in the blood circulation their glycan linkages are progressively hydrolyzed from the terminal position inward in what appears to be primarily a stepwise exo‐glycosidic process starting with neuraminidases and followed by a series of glycosidases that results in the exposure of different potential lectin ligands. We have also published that this mechanism includes cell surface glycoproteins of the intestinal tract. This intrinsic mechanism of glycoprotein aging is common to all plasma glycoproteins and appears to determine the half‐lives of glycoproteins in circulation in the limited cases where it has been studied ‐ specifically two circulating alkaline‐phosphatase enzymes. The presence of lectins that recognize further aged glycoproteins includes the mannose receptors, which would detect mannose ligands exposed following loss of sialic acid, galactose, and N‐acetylglucosamine linkages. Herein we have investigated the identities of glycoproteins modulated by mannose exposure with increasing age, and those are further regulated in their clearance and function by the endogenous macrophage mannose receptor (Mrc1). Mrc1 is found on macrophages and specialized cells in most organs as well as the vasculature and has a broad spectrum of both glycan and protein ligands due to its complex domain‐repeat structure composed of three distinct binding sites. A cysteine rich domain and a fibronectin like domain allow the binding of sulfated glycoproteins and collagens respectively. The lectin domain of Mrc1 can bind and endocytose mannosylated particles ranging from pathogens and allergens to mannosylated glycoproteins. Our studies reveal that the Mrc1 receptor has a lectin function that controls the half‐lives of specific glycoproteins in blood circulation, including those that have a regulatory role in cardiovascular and inflammatory processes.Support or Funding InformationNIH grant HL131474.

Anti-Alzheimer's Materials Isolated from Marine Bio-resources: A Review.

The most common type of dementia found in the elderly population is Alzheimer's disease. The disease not only impacts the patients and their families but also the society therefore, the main focus of researchers is to search new bioactive materials for treating AD. The marine environment is a rich source of functional ingredients and to date, we can find sufficient research relating to anti- Alzheimer's compounds isolated from marine environment. Therefore, this review focuses on the anti- Alzheimer's material from marine bio-resources and then expounds on the anti-Alzheimer's compounds from marine seaweed, marine animal and marine microorganisms. Moreover, because of the complexity of the disease, different hypothesizes have been elaborated and active compounds have been isolated to inhibit different stages of pathophysiological mechanisms. Sulfated polysaccharides, glycoprotein, and enzymatic hydrolysates from marine seaweeds, peptides, dietary omega-3 polyunsaturated fatty acids and skeletal polysaccharide from marine animals and secondary metabolites from marine microorganism are summarized in this review under the anti-Alzheimer's compounds from the marine.

Understanding the mechanism of copurification of "difficult to remove" host cell proteins in rituximab biosimilar products.

Host cell proteins (HCPs) are considered a critical quality attribute and are linked to safety and efficacy of biotherapeutic products. Researchers have identified 10 HCPs in Chinese hamster ovary (CHO) that exhibit common characteristics of product association, coelution, and age-dependent expression and therefore are "difficult to remove" during downstream purification. These include cathepsin D, clusterin, galectin-3-binding protein, G-protein coupled receptor 56, lipoprotein lipase, metalloproteinase inhibitor, nidogen-1 secreted protein acidic and rich in cysteine (SPARC), sulfated glycoprotein, and insulin-like growth factor-2 RNA-binding protein. While the levels of HCPs in the investigated biosimilars were within the acceptable range of <100 ppm, certain "difficult to remove" HCPs were found in the biosimilar samples. This article aims to elucidate the underlying interactions between these "difficult to remove" HCPs and the mAb product. Surface study of rituximab exhibited unstable discontinuous patches of residues on the protein surface that have high propensity to get buried and lower the solvent exposed area. The higher order structure and the receptor binding were not affected, except for one of the biosimilars, owing to extremely low-HCP levels in its final drug product. Finally, based on the combined experimental and computational data from this study, a probable mechanism of retention for the 10 HCPs is proposed. The results presented here can guide downstream process design or avenues for protein engineering during product discovery to achieve more effective removal of the impurities.

Serum Stem Cell Growth Factor Beta for the Prediction of Therapy Response in Hepatocellular Carcinoma.

Introduction Chronic inflammatory response is one of major contributors in the development of hepatocellular carcinoma (HCC). Inflammatory molecules, such as cytokines and growth factors in the circulation, can be useful in the diagnosis and prognosis of the patients. The stem cell growth factor beta (SCGFβ), a newly found protein, is a secreted sulfated glycoprotein and it functions as a growth factor for primitive hematopoietic progenitor cells. The level of SCGFβ had been reported to be elevated in several cancer types. However, there is very few or even no information on this protein in the study of HCC, even more in clinical studies. Methods A multiplex immunoassay panel of 48 cytokines and growth factors were utilized to screen 68 sera from 29 HCC patients at pretreatment (T0), 1 month (T1), and 6 months (T6) after treatment by either radiofrequency ablation (RF) or transarterial chemoembolization (TACE). Treatment response was evaluated according to mRECIST criteria. Results Immunoassay screening showed that the levels of IL-17, CTACK, TNFα, IL-2Rα, IL-8, and SCGFβ were different in Complete Responders (CR) and Nonresponders (NR) groups. At T0 and T1, the SCGFβ level was significantly the highest in NR (23.8 and 40.7 ng/mL, respectively), followed by early recurrence (25.4 and 25.0 ng/mL), and CR (6.7 and 5.3 ng/mL), independently from HCV, stages, and treatment type. Low basal SCGFβ level was associated with longer disease-free survival compared to high SCGFβ. Conclusion In this study, for the first time, we demonstrate that the high level of serum SCGFβ at pre- and posttreatment is associated with HCC nonresponsiveness.

Identification of glycoproteins in mucous cells of the gill epithelium of Colossoma macropomum after exposure to organophosphate

ABSTRACT The use of organophosphates has been recommended for fish, especially the trichlorfon to control parasites. Colossoma macropomum were exposed to trichlorfon during 96 hours and of total number of mucous cells decreased in the number of cells when compared to the control group. Glycoproteins acid, acid sulphated and neutral was identified in the gill epithelium. Neutra glycoprotein had a significant decrease between control and the sublethal concentration. Acid glycoprotein didn’t have any significant difference between the groups exposed to the trichlorfon, compared to the control group. Sulfated acidic glycoprotein in the group exposed to the trichlorfon was noticed a reduction in number of mucosal cells acidic sulphated. The differences between density cell and production glycoprotein was a response of these cells after exposure to xenobiotic. The reduction of neutral, acid and sulphated acid glycoprotein in the MC of the gill epithelium Colossoma macropomum may affect gills epithelial surface protection by reducing the formation of an unstirred layer and enhance the ion loss.

Trypanosoma cruzi serinecarboxipeptidase is a sulfated glycoprotein and a minor antigen in human Chagas disease infection.

In this work, the presence of sulfated N-glycans was studied in a high-mannose-type glycoprotein of Trypanosoma cruzi with serinecarboxipeptidase (TcSCP) activity. The immune cross-reactivity between purified SCP and Cruzipain (Cz) was evidenced using rabbit sera specific for both glycoproteins. Taking advantage that SCP co-purifies with Cz from Concanavalin-A affinity columns, the Cz-SCP mixture was desulfated, ascribing the cross-reactivity to the presence of sulfate groups in both molecules. Therefore, knowing that Cz is a sulfated glycoprotein, with antigenic sulfated epitopes (sulfotopes), SCP was excised from SDS-PAGE and the N-glycosydic chains were analyzed by UV-MALDI-TOF-MS, confirming the presence of short-sulfated high-mannose-type oligosaccharidic chains. Besides, the presence of sulfotopes was analyzed in lysates of the different parasite stages demonstrating that a band with apparent molecular weight similar to SCP was highly recognized in trypomastigotes. In addition, SCP was confronted with sera of infected people with different degrees of cardiac dysfunction. Although most sera recognized it in different groups, no statistical association was found between sera antibodies specific for SCP and the severity of the disease. In summary, our findings demonstrate (1) the presence of sulfate groups in the N-glycosidic short chains of native TcSCP, (2) the existence of immune cross-reactivity between Cz and SCP, purified from epimastigotes, (3) the presence of common sulfotopes between both parasite glycoproteins, and (4) the enhanced presence of sulfotopes in trypomastigotes, probably involved in parasite-host relationship and/or infection. Interestingly, we show for the first time that SCP is a minor antigen recognized by most of chronic Chagas disease patient's sera.

Sequential activation of the AKT pathway in human osteoblasts treated with Oscarvit: a bioactive product with positive effect both on skeletal pain and mineralization in osteoblasts

BackgroundOscarvit (OSC) is an in-house preparation consisting of calcium, magnesium, phosphorus, strontium, Vitamin D, and eggshell membrane hydrolysate containing naturally occurring glycosaminoglycans and sulfated glycoproteins. OSC has been used both in an open-label human study and in vitro in osteoblasts.MethodsFifteen patients divided into three groups received oral OSC 0.6 g three times daily for 20 days. The main outcome measures were regional skeletal pain over the treatment period. For the in vitro experiments eight primary human osteoblasts cultures were established from trabecular bone, six of them from the femoral head, and two from the tibia. Cells were cultured for five to 20 days in the presence of 20 μg/ml OSC. Immunocytochemistry and RT-PCR were used to detect molecular alterations involved in the mineralization process. Calcium concentration was measured by means of a colorimetric assay and cell viability was analyzed using the LDH cytotoxicity assay. To investigate whether the osteoblasts response to OSC is associated with signaling processes the ERK1/2 and AKT signal transduction pathways were analyzed.ResultsOpen label human study: OSC, 0.6 g three times daily, resulted in a significant positive effect on pain alleviation of 42% after 5 days (p < 0.001), 57% after 10 days and 68% after 20 days (p < 0.0001; for both time points), with no side-effects being reported. In vitro analysis: In osteoblasts, growing in OSC-supplemented media significant overexpression of bone γ-carboxylglutamic acid-containing protein, secreted phosphoprotein-1, integrin binding sialoprotein, and dentin matrix phosphoprotein genes could be detected when compared to control osteoblasts grown in the absence of OSC. Moreover, OSC-treated osteoblasts produced over the study period vast extracellular calcium deposits without any loss of cellular integrity or signs of cellular toxicity. In addition OSC promotes osteoblast differentiation and activates the AKT signaling pathway.ConclusionThis open label study provides preliminary evidence of the efficacy of OSC. Despite the limitations (small heterogeneous patient group) the findings can be viewed as a necessary investigation that supports further clinical trials with a double-blind controlled design. Experiments at cellular and molecular level provide supplementary information about OSC that increases mineralization in osteoblasts and activation of the AKT pathway.Trial registrationDRKS00013233, 06th November 2017, retrospectively registered.

Specific Unbinding Forces Between Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 Measured Using Force Spectroscopy.

Protein tyrosine sulfation (PTS) is a key modulator of extracellular protein-protein interaction (PPI), which regulates principal biological processes. For example, the capsid protein VP1 of enterovirus 71 (EV71) specifically interacts with sulfated P-selectin glycoprotein ligand-1 (PSGL-1) to facilitate virus invasion. Currently available methods cannot be used to directly observe PTS-induced PPI. In this study, atomic force microscopy was used to measure the interaction between sulfated or mutated PSGL-1 and VP1. We found that the binding strength increased by 6.7-fold following PTS treatment on PSGL-1 with a specific antisulfotyrosine antibody. Similar results were obtained when the antisulfotyrosine antibody was replaced with the VP1 protein of EV71; however, the interaction forces of VP1 were only approximately one-third of those of the antisulfotyrosine antibody. We also found that PTS on the tyrosine-51 residue of glutathione S-transferases fusion-PSGL-1 was mainly responsible for the PTS-induced PPI. Our results contribute to the fundamental understanding of PPI regulated through PTS.