Crosslinking of pellicle precursor proteins by oral transglutaminase
Date
1999
DOI
Authors
Yao, Yuan
Version
OA Version
Citation
Abstract
Acidic proline-rich proteins (PRPs), statherin and histatins have been demonstrated to be the major components in the acquired enamel pellicle. Recent studies have identified several types of protein complexes in the oral cavity involving covalent and noncovalent interactions. Among these interactions, the transglutaminase catalyzed crosslinking of a glutamine residue with a lysine residue forms a [gamma]-glutamyl-[epsilon]-lysine covalent bond. This reaction may render protein substrates insoluble. Therefore, this process is likely to be one mechanism for the formation of the acquired enamel pellicle. Characterization of transglutaminase-catalyzed reaction with pellicle precursor proteins was investigated in this study with two ultimate aims. The first aim was to determine which pellicle precursor proteins can act as substrates for transglutaminase, and the second aim was to isolate and characterize a crosslinked protein formed between two purified pellicle precursor proteins with transglutaminase obtained from buccal epithelial cells. To investigate the first aim, dansyl cadaverine was used to study the reactivity of glutamine residues in acidic large and small proline-rich proteins, statherin, and the major histatins, whereas a glutamine containing dansylated peptide was used to study the reactivity of lysine residues. Incorporation of either of these dansylated molecules into a pellicle precursor protein by transglutaminase was qualitatively assessed by gel electrophoresis. Quantitative measurements of reactive glutamine and lysine residues revealed that almost all of the lysines present in the acidic PRPs and statherin, half of the lysines present in histatin 1, and a small percentage of lysine in histatin 3 and histatin 5 could participate in the crosslink reaction. Glutamine reactivity was also detected, but at a lower percentage of available residues ranging from 1% for small PRPs to 14% for statherin. These results demonstrate that primary pellicle precursor proteins, acidic proline-rich proteins, statherin, and the major histatins are capable of undergoing crosslinking reactions induced by oral transglutaninase. To address the second aim, acidic PPR-1 and statherin were incubated with transglutaminase and the crosslinked product was characterized. SDS- -PAGE revealed a new protein band with the apparent molecular weight of 32 kDa, consistent with the combined apparent molecular weights of PRP-1 or Statherin. HPLC analysis revealed an additional peak. Amino acid analysis of the material contained in this peak indicated that it contained PRP-1 and statherin. Furthermore, the existence of a covalent [gamma]-glutanyl-[epsilon]-lysine crosslink was confirmed by amino acid analysis of this protein after pronase digestion. Additional verification of the existence of [gamma]-glutamine-[epsilon]-lysine was achieved using TNBS (trinitrobenzosulfonic acid) to label the crosslinked protein. Lysine was only detected from acid hydrolyzed TBNS-labeled protein and not from pronase digested TNBS-labeled protein. This suggested that the lysine released after acid hydrolysis was from the crosslinked dipeptide, [gamma]-glutamyl-[epsilon]-lysine. The data obtained indicated that oral epithelial cell derived transglutaminase is capable of crosslinking pellicle precursor proteins. In addition, this finding exemplifies the potential of post-secretory processing of salivary proteins, which may generate new protein species with additional functional capacities.
Description
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please log in with a valid BU account to access and click Download. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.
Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1999 (Oral Biology).
Includes bibliographical references (leaves 120-143).
Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1999 (Oral Biology).
Includes bibliographical references (leaves 120-143).
License
This work is protected by copyright. Downloading is restricted to the BU community. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.