Biology subjects

Sobala, L. F.

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

4 recordsLinked to original sources

bioRxiv2025

Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation

Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: Trichoplusia ni insect cells (High Five) and human embryonic kidney cells (Expi293F). The High Five cells generate short oligomannose structures, while the Expi293F cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts. Highlights- Active recombinant Gb3/CD77 synthase was obtained in High Five and Expi293F cells. - High Five cells provided higher recombinant Gb3/CD77 synthase yield than Expi293F. - Glycosylation pattern of recombinant Gb3/CD77 synthase was host specific. - Activity of Expi293F-derived enzyme depended on 6xHis-tag localization.

biochemistry↗

bioRxiv2024

LukProt: A database of eukaryotic predicted proteins designed for investigations of animal origins

AO_SCPLOWBSTRACTC_SCPLOWThe origins and early evolution of animals is a subject with many outstanding questions. One problem faced by researchers trying to answer them is the absence of a comprehensive database with sequences from non-bilaterians. Publicly available data is plentiful but scattered and often not associated with proper metadata. A new database presented in this paper, LukProt, is an attempt at solving this issue. The database contains protein sequences obtained mostly from genomic, transcriptomic and metagenomic studies and is an extension of EukProt (Richter et al., 2022, Peer Community Journal, 2, e56). LukProt adopts the EukProt naming conventions and includes data from 216 additional animals. The database is associated with a taxonomic grouping (taxogroup) scheme suitable for studying early animal evolution. Minor updates to the database will contain species additions or metadata corrections and major updates will synchronize LukProt to each new version of EukProt and releases are permanently stored on Zenodo. A BLAST server to search the database is available at https://lukprot.hirszfeld.pl/. Users are invited to participate in maintaining and correcting LukProt. As it can be searched without downloading locally, the database can be a convenient resource not only for evolutionary biologists, but for the broader scientific community as well. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/577650v4_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@14b37c7org.highwire.dtl.DTLVardef@13b010corg.highwire.dtl.DTLVardef@c1cdecorg.highwire.dtl.DTLVardef@1b66438_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementLukProt is a sequence database aiming to accelerate the research on the evolution of animals by cutting the time-consuming step of assembling sequences from disparate sources. Non-bilaterians are currently not well covered by general purpose databases, despite plentiful, public sequencing data. These data were integrated into a consistently curated database, presented here. It can be downloaded and used locally or used via a public BLAST search server. A clear taxonomic framework is also introduced, as well as scripts to aid local data analyses. LukProt will be publicly available on Zenodo, kept up to date and synchronized with each new version of its parent database, EukProt.

bioinformatics↗

bioRxiv2022

Evolution and phylogenetic distribution of endo-α-mannosidase

While glycans underlie many biological processes, such as protein folding, cell adhesion and cell-cell recognition, deep evolution of glycosylation machinery remains an understudied topic. N-linked glycosylation is a conserved process in which mannosidases are key trimming enzymes. One of them is the glycoprotein endo--1,2-mannosidase which participates in the initial trimming of mannose moieties from an N-linked glycan inside the cis-Golgi. It is unique as the only endo-acting mannosidase found in this organelle. Relatively little is known about its origins and evolutionary history; so far it was thought to occur only in vertebrates. Here I perform a taxon-rich bioinformatic survey to unravel the evolutionary history of this enzyme, including all major eukaryotic clades and a wide representation of animals. I found the endomannosidase to be vastly more widely distributed in animals than previously thought and in fact present in almost all eukaryotic clades. I tracked protein motif changes in context of the canonical animal enzyme. Additionally, my data show that the two canonical versions of endomannosidase in vertebrates, MANEA and MANEAL, arose at the second round of the two vertebrate genome duplications and indicate presence of a third protein, named here CMANEAL. Finally, I describe a framework where N-glycosylation co-evolved with complex multicellularity. A better understanding of the evolution of core glycosylation pathways is pivotal to understanding biology of eukaryotes in general, and the Golgi apparatus in particular. This systematic analysis of the endomannosidase evolution is one step towards this goal.

evolutionary biology↗

bioRxiv2020

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo--1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and unusually is located within the Golgi, where it allows ER escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N-glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation. SIGNIFICANCE STATEMENTThe glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo--1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.

biochemistry↗