Loss of core-fucosylation of SPARC impairs collagen binding and contributes to COPD.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with high morbidity and mortality worldwide. Although several mechanisms to account for deleterious immune effects were proposed, molecular description for the underlying alveolar structural alterations for COPD is lacking. Here, silencing of α1,6-fucosyltransferase (Fut8), the enzyme for core-fucosylation and highly expressed in lung stem cells, resulted in alveolar structural changes in lung organoids, recapitulating COPD. Site-specific mass spectrometry analysis demonstrated that the secreted protein acidic and rich in cysteine (SPARC), which binds collagen, contains a core-fucosylation site in its VCSNDNcfK glycopeptide. Biacore assay showed markedly reduced collagen binding of SPARC lacking core fucosylation. Molecular dynamics analysis revealed that core fucosylation of SPARC-induced dynamic conformational changes in its N-glycan, allowing terminal galactose and N-acetylglucosamine to interact with K150, P261 and H264 residues, thereby promoting collagen binding. Site-specific mutagenesis of these residues also resulted in low affinity for collagen binding. Moreover, loss of collagen and decline of core fucosylation were observed in COPD lung tissues. These findings provide a new mechanistic insight into the role of core fucosylation of SPARC in cell-matrix communication and contribution to the abnormal alveolar structures in COPD.

Sample Size-Comparable Spectral Library Enhances Data-Independent Acquisition-Based Proteome Coverage of Low-Input Cells.

Despite advancements of data-independent acquisition mass spectrometry (DIA-MS) to provide comprehensive and reproducible proteome profiling, its utility in very low-input samples is limited. Due to different proteome complexities and corresponding peptide ion abundances, the conventional LC-MS/MS acquisition and widely used large-scale DIA libraries may not be suitable for the micro-nanogram samples. In this study, we report a sample size-comparable library-based DIA approach to enhance the proteome coverage of low-input nanoscale samples (i.e., nanogram cells, ∼5-50 cells). By constructing sample size-comparable libraries, 2380 and 3586 protein groups were identified from as low as 0.75 (∼5 cells) and 1.5 ng (∼10 cells), respectively, highlighting one of the highest proteome coverage with good reproducibility (86%-99% in triplicate results). For the 0.75 ng sample (∼5 cells), significantly superior identification (2380 proteins) was achieved by small-size library-based DIA, compared to 1908, 1749, and 107 proteins identified from medium-size and large-size libraries and a lung cancer resource spectral library, respectively. A similar trend was observed using a different instrument and data analysis pipeline, indicating the generalized conclusion of the approach. Furthermore, the small-size library uniquely identified 518 (22%) proteins in the low-abundant region and spans over a 5-order dynamic range. Spectral similarity analysis revealed that the fragmentation ion pattern in the DIA-MS/MS spectra of the dataset and spectral library play crucial roles for mapping low abundant proteins. With these spectral libraries made freely available, the optimized library-based DIA strategy and DIA digital map will advance quantitative proteomics applications for mass-limited samples.

Sialylation of vasorin by ST3Gal1 facilitates TGF-ß1-mediated tumor angiogenesis and progression.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-ß1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-ß1 by 2- to 3-fold and thereby dampening TGF-ß1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-ß1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-ß1 upregulates ST3Gal1 to circumvent the negative impact of VASN.

Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation.

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry.

Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications.