Beck, M. R. | bioRxiv Science

bioRxiv2020

Conserved tryptophan mutation disrupts structure and function of immunoglobulin domain revealing unusual tyrosine fluorescence

Immunoglobulin (Ig) domains are the most prevalent protein domain structure and share a highly conserved folding pattern; however, this structural family of proteins is also the most diverse in terms of biological roles and tissue expression. Ig domains vary significantly in amino acid sequence but share a highly conserved tryptophan in the hydrophobic core of this beta-stranded protein. Palladin is an actin binding and bundling protein that has five Ig domains and plays an important role in normal cell adhesion and motility. Mutation of the core tryptophan in one Ig domain of palladin has been identified in a pancreatic cancer cell line, suggesting a crucial role for this sole tryptophan in palladin Ig domain structure, stability, and function. We found that actin binding and bundling was not completely abolished with removal of this tryptophan despite a partially unfolded structure and significantly reduced stability of the mutant Ig domain as shown by circular dichroism investigations. In addition, this mutant palladin domain displays a tryptophan-like fluorescence attributed to an anomalous tyrosine emission at 345 nm. Our results indicate that this emission originates from a tyrosinate that may be formed in the excited ground state by proton transfer to a nearby glutamyl residue. Furthermore, this study emphasizes the importance of tryptophan in protein structural stability and illustrates how tyrosinate emission contributions may be overlooked during the interpretation of the fluorescence properties of proteins. SHORT ABSTRACTThis study explores the functional and structural consequences of a point mutation in palladin, an Ig domain protein first identified in a pancreatic tumor cancer cell line. While exploring the consequences of mutating this conserved tryptophan in the hydrophobic core of the most prevalent domain structure found in proteins, an anomalous tyrosine fluorescence phenomenon was exposed.

biochemistry↗

bioRxiv2020

IDENTIFICATION AND CHARACTERIZATION OF A LARGE SOURCE OF PRIMARY MESENCHYMAL STEM CELLS TIGHTLY ADHERED TO BONE SURFACES OF HUMAN VERTEBRAL BODY MARROW CAVITIES

Therapeutic allogeneic mesenchymal stem/stromal cells (MSC) are currently in clinical trials for evaluating their effectiveness in treating many different disease indications. Eventual commercialization for broad distribution will require further improvements in manufacturing processes to economically manufacture MSC at sufficient scales required to satisfy projected demands. A key contributor to the present high cost of goods (COG) for MSC manufacturing is the need to create master cell banks (MCBs) from multiple donors, which leads to variability in large-scale manufacturing runs. Therefore, the availability of large single donor depots of primary MSC would greatly benefit the cell therapy market by reducing costs associated with manufacturing. We have discovered that an abundant population of cells possessing all the hallmarks of MSC is tightly associated with the vertebral body (VB) bone matrix and are only liberated by proteolytic digestion. Here we demonstrate that these vertebral bone-adherent (vBA) MSC possess all the International Society of Cell and Gene Therapy (ISCT)-defined characteristics (e.g., plastic adherence, surface marker expression, and trilineage differentiation) of MSC and, therefore, have termed them vBA-MSC, to distinguish this population from loosely associated MSC recovered through aspiration or rinsing of the bone marrow (BM) compartment. Pilot banking and expansion was performed with vBA-MSC obtained from 3 deceased donors and it was demonstrated that bank sizes averaging 2.9x108 {+/-} 1.35x108 vBA-MSC at passage one were obtainable from only 5 g of digested VB bone fragments. Each bank of cells demonstrated robust proliferation through a total of 9 passages without significant reduction in population doubling times. The theoretical total cell yield from the entire amount of bone fragments (approximately 300g) from each donor with limited expansion through 4 passages is 100 trillion (1x1014) vBA-MSC, equating to over 105 doses at 10x106 cells/kg for an average 70 kg recipient. Thus, we have established a novel and plentiful source of MSC which will benefit the cell therapy market by overcoming manufacturing and regulatory inefficiencies due to donor-to-donor variability.

bioengineering↗