In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

O-glycan initiation directs distinct biological pathways and controls epithelial differentiation.

Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc-Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets with cell-ECM adhesion, and GalNAc-T3 targets with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.

Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Characterization of TGF-ß signaling in a human organotypic skin model reveals that loss of TGF-ßRII induces invasive tissue growth.

Transforming growth factor-ß (TGF-ß) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-ß signaling pathway, including the ligands TGF-ß1, TGF-ß2, and TGF-ß3, the receptor TGF-ßRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-ß signaling, the loss of TGF-ß1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-ßRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-ßRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-ßRII. This study provides a framework for exploring TGF-ß signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models.

Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids. For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020).

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication.

In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside transporter human equilibrative nucleoside transporter 1 (hENT1) and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1-in addition to repressing dNTP synthesis-together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.

Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.