Using targeted proteomics-based detection of collagen propeptides to quantify fibrillar collagen biogenesis in vitro.

The collagen superfamily, as the major structural component of the extracellular matrix, encompasses 28 distinct subtypes, with type-I and -III forming fibrils crucial for the matrix scaffold. During collagen biogenesis, trimers of type-I and -III procollagen are secreted into the extracellular matrix. The N- and C-terminal propeptides of these trimers are proteolytically cleaved from procollagen during secretion, initiating collagen fibril formation. The propeptides are released into extracellular space and, therefore, have been used to quantify collagen biogenesis. But high-throughput methods for the quantification of these biomarkers are still lacking. This study presents a state-of-the-art multiplexed approach for the simultaneous quantification of PINP, PICP, PIIINP and PIIICP from cell culture supernatants. The ability of targeted proteomics to quantify these propeptides from cell culture samples was assessed in this study. Using tryptic digestion and solid phase extraction, we were able to accurately quantify precollagen propeptides in a range of 3-1000 ng/mL. The assay showed an average inter-assay variance of 6.86 % with an overall recovery ranging from 92 to 98 %. The assay was validated using recombinant protein standards diluted in surrogate matrix and tested using transforming growth factor ß1 mediated induction of normal human dermal fibroblasts. In summary, the assay presented in this paper offers a novel, robust, and precise high-throughput method for measuring human collagen propeptides in cell culture supernatants, empowering researchers to assess collagen biogenesis effectively in in vitro experiments.

Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction.

Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFß1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

A novel SPE-UPLC-MS/MS-based assay for the selective, simultaneous quantification of xylosyltransferase-I and -II activity.

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan. The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results. Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.

Enzymatic Peptide and Protein Bromination: The BromoTrp Tag.

Bio-orthogonal reactions for modification of proteins and unprotected peptides are of high value in chemical biology. The combination of enzymatic halogenation with transition metal-catalyzed cross-coupling provides a feasible approach for the modification of proteins and unprotected peptides. By a semirational protein engineering approach, variants of the tryptophan 6-halogenase Thal were identified that enable efficient bromination of peptides with a C-terminal tryptophan residue. The substrate scope was explored using di-, tri-, and tetrapeptide arrays, leading to the identification of an optimized peptide tag we named BromoTrp tag. This tag was introduced into three model proteins. Preparative scale post-translational bromination was possible with only a single cultivation and purification step using the brominating E. coli coexpression system Brocoli. Palladium-catalyzed Suzuki-Miyaura cross-coupling of the bromoarene was achieved with Pd nanoparticle catalysts at 37 °C, highlighting the rich potential of this strategy for bio-orthogonal functionalization and conjugation.