Biology subjects

Zacchi, L. F.

Publications and source records attributed to Zacchi, L. F..

8 recordsLinked to original sources

bioRxiv2024

Isolation and Characterization of Antibodies Against VCAM-1 Reveals Putative Role for Ig-like Domains 2 and 3 in Cell-to-Cell Interaction

Vascular cell adhesion molecule-1 (VCAM-1) plays an important role in inflammation, where it facilitates the recruitment of leukocytes to the inflamed area via leukocytes VLA-4 and endothelial cells VCAM-1 interaction. VCAM-1 expression is also upregulated in certain cancers. VCAM-1 has 7 Ig-like domains, with domains 1 and 4 shown to be critical for VLA-4 binding. However, the specific functions of individual VCAM-1 Ig-like domains remain poorly understood. In this study, we identified single-chain variable fragment (scFvs) antibodies targeting domains 2, 3, and 5 of VCAM-1, and investigated the ability of these antibodies to block VCAM-1-mediated cell adhesion to macrophages. We show that scFv antibodies against Ig-like domains 2 and 3 significantly interfere with the ability of macrophages to bind endothelial cells, suggesting that these domains also play a role in facilitating this interaction. These results emphasize the need to more carefully study the role of each domain on VCAM-1 function and highlight the potential of targeting these VCAM-1 domains for more tailored therapeutic interventions in inflammatory diseases and cancer.

molecular biology↗

bioRxiv2023

Tor1 dysfunction promotes phenotypic diversity in the asexual fungus Candida albicans

Genetic variation is a primary contributor to phenotypic variation within a population. In asexual eukaryotes however, it is unclear how, or if, genetic variation is generated and maintained to promote phenotypic variation. C. albicans, an asexual fungus that causes opportunistic infections in susceptible hosts, has several phenotypic switching systems, including the colony morphology phenotypic switching (CMPS) system. CMPS, a penetrant change in colony morphology on solid medium in vitro, is associated with incipient or fulminant clinical disease. CMPS results in the alteration of additional virulence properties, including drug resistance, that are not tightly correlated with colony morphology. Importantly, it is unknown whether CMPS is a regulated or stochastic process. We found that specific mutants affecting the Target of Rapamycin (TOR) pathway showed an increase in CMPS frequency and CMPS in these mutant backgrounds was associated with changes in rapamycin sensitivity. We also identified growth conditions that promoted CMPS in clinical strains and found that CMPS in these backgrounds was also linked to the TOR pathway through changes in rapamycin sensitivity. These results demonstrate that CMPS promotes phenotypic variation through the TOR pathway, supporting a model that this is a regulated process. Since the TOR growth control pathway is conserved throughout the eukarya, the identification of TOR as a phenotypic diversity regulator likely has broad implications.

genetics↗

bioRxiv2021

N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251{+/-}70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

biochemistry↗

bioRxiv2020

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete {gamma}-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX post-translational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, {gamma}-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.

biochemistry↗

bioRxiv2020

S-Trap eliminates cell culture media polymeric surfactants for effective proteomic analysis of mammalian cell bioreactor supernatants

Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust clean-up and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published datasets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

biochemistry↗

bioRxiv2019

Glycoproteomic measurement of site-specific polysialylation

Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/740928v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@928857org.highwire.dtl.DTLVardef@353842org.highwire.dtl.DTLVardef@a97aorg.highwire.dtl.DTLVardef@176a134_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biochemistry↗

bioRxiv2019

DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides

Data Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest. These DIA ion libraries can be theoretical or built from peptide identification data from Data Dependent Acquisition (DDA) MS workflows. However, DDA libraries derived from empirical data rely on confident peptide identification, which can be challenging for peptides carrying complex post-translational modifications. Here, we present DIALib, software to automate the construction of peptide and glycopeptide Data Independent Acquisition ion Libraries. We show that DIALib theoretical ion libraries can identify and measure diverse N- and O-glycopeptides from yeast and mammalian glycoproteins without prior knowledge of the glycan structures present. We present proof-of-principle data from a moderately complex yeast cell wall glycoproteome and a simple mixture of mammalian glycoproteins. We also show that DIALib libraries consisting only of glycan oxonium ions can quickly and easily provide a global compositional glycosylation profile of the detectable "oxoniome" of glycoproteomes. DIALib will help enable DIA glycoproteomics as a complementary analytical approach to DDA glycoproteomics.

bioinformatics↗

bioRxiv2019

Identification of novel glycosylation events on human serum-derived Factor IX

Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

biochemistry↗