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Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To improve breast radiographers' individual performance by using automated software to assess the correctness of breast positioning and compression in tomosynthesis screening., Materials and Methods: In this retrospective longitudinal analysis of prospective cohorts, six breast radiographers with varying experience in the field were asked to use automated software to improve their performance in breast compression and positioning. The software tool automatically analyzes craniocaudal (CC) and mediolateral oblique (MLO) views for their positioning quality by scoring them according to PGMI classifications (perfect, good, moderate, inadequate) and checking whether the compression pressure is within the target range. The positioning and compression data from the studies acquired before the start of the project were used as individual baselines, while the data obtained after the training were used to test whether conscious use of the software could help the radiographers improve their performance. The percentage of views rated perfect or good and the percentage of views in target compression were used as overall metrics to assess changes in performance., Results: Following the use of the software, all radiographers significantly increased the percentage of images rated as perfect or good in both CCs and MLOs. Individual improvements ranged from 7 to 14% for CC and 10 to 16% for MLO views. Moreover, most radiographers exhibited improved compression performance in CCs, with improvements up to 16%., Conclusion: Active use of a software tool to automatically assess the correctness of breast compression and positioning in breast cancer screening can improve the performance of radiographers., Clinical Relevance Statement: This study suggests that the use of a software tool for automatically evaluating correctness of breast compression and positioning in breast cancer screening can improve the performance of radiographers on these metrics, which may ultimately lead to improved screening outcomes., Key Points: • Proper breast positioning and compression are critical in breast cancer screening to ensure accurate diagnosis. • Active use of the software increased the quality of craniocaudal and mediolateral oblique views acquired by all radiographers. • Improved performance of radiographers is expected to improve screening outcomes., (© 2023. The Author(s).)

We report on laser cooling of a large fraction of positronium (Ps) in free flight by strongly saturating the 1^{3}S-2^{3}P transition with a broadband, long-pulsed 243 nm alexandrite laser. The ground state Ps cloud is produced in a magnetic and electric field-free environment. We observe two different laser-induced effects. The first effect is an increase in the number of atoms in the ground state after the time Ps has spent in the long-lived 2^{3}P states. The second effect is one-dimensional Doppler cooling of Ps, reducing the cloud's temperature from 380(20) to 170(20) K. We demonstrate a 58(9)% increase in the fraction of Ps atoms with v&#95;{1D}<3.7×10^{4}  ms^{-1}.

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy., Competing Interests: A patent application has been filed by the University of Copenhagen. GlycoDisplay ApS has license rights to the patent application. ZY, WT, CK, and HC are named co-inventors, and ZY, CK, and HC have financial interests in GlycoDisplay ApS. Y-HC is an employee of GlycoDisplay ApS. RD is a Consultant to Genzyme-Sanofi and Sangamo Therapeutics, Inc. He owns founder stock in Amicus Therapeutics and options for Sangmo Therapeutics, Inc. and receives royalities from Genzyme-Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Tian, Yasuda, Ye, Song, Mandel, Kristensen, Povolo, Marques, Čaval, Heck, Sampaio, Johannes, Tsukimura, Desnick, Vakhrushev, Yang and Clausen.)

Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.