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All Subjects: Pleiotrophin
Creators: Wang, Xu

Expression of Pleiotrophin as Separate Domains to Examine Glycosaminoglycan Binding Sites

Description

Glycosaminoglycan (GAG) binding by the cytokine pleiotrophin (PTN) was examined by expressing both thrombospondin 1 type-1 repeat domains of PTN separately, as PTN-N and PTN-C. PTN-N contains residues 31-89, and PTN-C contains residues 90-146. Nuclear magnetic resonance (NMR) experiments were conducted on both PTN-N and PTN-C to elucidate GAG binding regions. Titration with heparin dp6 showed a twofold increase in affinity when expressing PTN-N and PTN-C separately rather than as intact PTN. Paramagnetic relaxation rate enhancement experiments and surface paramagnetic relaxation enhancement (PRE) perturbation experiments were used to determine which residues were involved in GAG binding. One binding site was observed in PTN-N, around residue T82, and two binding sites were observed in PTN-C, one around residue K93 and the other around residue G142. These observed binding sites agree with the binding sites already proposed by the Wang lab group and other studies. Future work on the subject could be done on confirming that other varieties and length GAGs bind at the same sites, as well as examining the effect longer GAG fragments have on the affinity of intact PTN versus separate domains.

ContributorsKuch, Nathaniel Jacob (Author) / Wang, Xu (Thesis director) / Van Horn, Wade (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2015-12

Producing Glycosaminoglycan-Binding Proteins Containing Single-Isotope-Labeled Lysine as a Tool to Study Protein-Glycosaminoglycan Interactions

Description

Pleiotrophin (PTN) is a cell-signaling protein in the human body that plays a pivotal role in the development of the central nervous system. It is known to have a high affinity for glycosaminoglycan (GAG), a type of linear polysaccharide. PTN has the ability to bind to a wide range of receptors, including receptor-type protein tyrosine phosphatase ζ (PTPRZ), a protein expressed in embryonic stem cells that regulates signals associated with survival, cell proliferation, and stem cell pluripotency. Several of these receptors are proteoglycans that carry GAGs, and the interaction between PTN and GAG has proven to be crucial to PTN’s functionality. Though PTN performs several important biochemical duties in normal cellular processes, this protein is upregulated in various cancer cell lines, primarily glioblastoma, an aggressive form of cancer that arises in the brain or spinal cord. The high levels of PTN expression in these forms of cancer may correlate to the cancer cells’ metastatic ability in the body. Determining how these PTN-GAG interactions form in cells is imperative for understanding how they may correlate to the development of cancer cell lines such as glioblastoma. However, due to the NMR signal degeneracy among the lysines in PTN, it is currently not possible to distinguish between lysines that have strong interactions with GAG and those that do not. To overcome this, pyrrolysyl-tRNA synthetase-mediated amber codon suppression is used to incorporate a single 15N-labeled lysine, Boc-lysine (Boc-K), at a specific position. This thesis seeks to optimize the systems and conditions needed to achieve amber codon suppression. The Origami B (DE3) strain is commonly used to achieve this, and demonstrates positive expression of PTN. The first aim of this project is to determine whether SHuffle® demonstrates enhanced expression of PTN and, therefore, incorporation of Boc-K. However, upon comparing PTN expression results, it was found that SHuffle® and Origami B(DE3) demonstrated similar levels of PTN expression. This project's second phase is focused on using C321.ΔA (Church) strain to evaluate differences in PTN expression compared to SHuffle® and Origami B(DE3). Expression testing indicated, however, that the expression of PTN in Church strain was inconclusive.

ContributorsKuchibhotla, Aditya (Author) / Wang, Xu (Thesis director) / Mills, Jeremy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)

Created2023-05

αMI-domain of Integrin Mac-1 Binds the Cytokine Pleiotrophin Using Multiple Mechanisms

Description

The integrin Mac-1 (αMβ2, CD11b/CD18) is an important adhesion receptorexpressed on macrophages and neutrophils. It plays a crucial role in phagocytosis, cell-cell fusion, and cell migration. αMβ2 is also the most promiscuous integrin with over 100 known ligands that span a broad range of physical and chemical attributes, many of which bind to the inserted (I) domain from the αM subunit. The interaction of αMI-domain with cytokine pleiotrophin (PTN) were determine. PTN is a cationic protein known to induce Mac-1- mediated adhesion and migration in cells. The data showed that PTN’s interaction with αMI-domain contains both divalent cation-dependent and independent mechanisms. In particular, PTN’s N-terminal domain has weak interactions with the N/C-termini side of αMI-domain using a metal-independent mechanism. However, stronger interaction is achieved through the chelation of the divalent cation in the metal ion-dependent adhesion site of active αMI-domain by PTN’s acidic residues. Although many acidic residues in PTN can act as the chelator, active αMI-domain’s interaction with PTN’s E98 plays an especially important role. NOE, chemical shift perturbation (CSP) data, and mutagenesis studies showed residues near E98 are at the binding interface and the E98 mutation greatly reduced binding affinity between two proteins. Interestingly, the CSP and MD simulation data showed the binding interface can be supported by the interaction of PTN’s H95 with the acidic clusters D242, E244, and D273 from αMI-domain, while PTN’s E66 form electrostatic interaction with R208 and K245 from αMI-domain. The determined recognition motif of αMI-domain for its ligands is (H/R/K)xxE. The ability to accommodate the longer distance between E and (H, R, K) compared to the zwitterionic motif RGDii explained how αMβ2 can interact with a large repertoire of ligands and be versatile in its functional portfolio.

ContributorsNguyen, Hoa Thi Thanh (Author) / Wang, Xu (Thesis advisor) / Van Horn, Wade (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)

Created2024