Soo Il Chung

Crosslinking of uteroglobin by transglutaminase

Uteroglobin, a progesterone induced, pregnancy related protein, can be incorporated into higher molecular weight proteins by human placental Factor XIIIa. This process is time dependent, requires CaCl2 and can be inhibited by the addition of polylysine, dansylcadavarine or histamine. Crosslinking of uteroglobin into higher molecular weight proteins can also be brought about by guinea pig liver transglutaminase. Such a process may be involved in the modification of epididymal spermatozoa to suppress their antigenicity.

Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain.

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.

MULTIPLE MOLECULAR FORMS OF TRANSGLUTAMINASES IN HUMAN AND GUINEA PIG

ABSTRACT. The multiple molecular forms of transglutaminases in human and guinea pig may be classified into three distinct groups on the basis of physical, chemical, immunological, and enzymatic properties. These enzymes are: 1) the protransglutaminases (Factor XIII) of plasma, platelet, placenta, prostate gland, uterus, and liver, 2) the tissue transglutaminase of various organs and tissues, 3) the hair follicle transglutaminase. Each transglutaminase catalyzes a simple acyl-transfer reaction by a common catalytic mechanism (Folk, 1969). Tissue and plasma transglutaminases appear to have a common spatial arrangement within their glutamine side chain binding sites. However, each enzyme displays pronounced differences in its catalytic efficiency toward the various peptide substrates. Each of the transglutaminases plays a specified role in its respective physiological processes although the exact role of the tissue enzyme is not yet certain.

Paradoxical effects of elastase inhibitor guamerin on the tissue repair of two different wound models: sealed cutaneous and exposed tongue wounds

Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.

Transient Expression of Transglutaminase C During Prenatal Development of Human Muscles

Tissue transglutaminase (TGase C, TGase II) is known to participate in cellular processes during morphogenesis, differentiation, and development of various prenatal tissues and organs. The expression of TGase C during myoblast proliferation and attachment to external laminae was examined by immunohistochemical (IH) localization at 5–12 weeks of developmental stages of prenatal human muscle in 23 embryos. IH detection using a monospecific antibody to TGase C showed a prominent expression of TGase C in muscle cells as stage- and spatial-specific patterns during an early embryonal period. The myoblasts of intervertebral, tongue, and limb muscles, attached to adjacent cartilaginous skeletons or fibrous fascia, showed a pronounced expression of TGase C at 5–6, 6–7, and 7–8 weeks after fertilization, respectively. The most intense activity of TGase C was observed in some cardiac myoblasts infiltrating into endocardial mesenchyme at 6–7 weeks after fertilization. Although weak staining was detected until 14 weeks after fertilization, the level of TGase C expression in all muscles was significantly decreased after 6–7 weeks, with the exception that the smooth muscle cells of blood vessels and gastrointestinal tract showed diffusely intense staining of TGase C between 5 and 12 weeks after fertilization. Western blotting analysis of the cellular extracts of pooled samples showed a single strong band at 80 kD at 6 weeks after fertilization. This band became weaker after 8–10 weeks of prenatal development. These findings of transient expression of TGase C, which coincides with the development of myoblast anchoring and differentiation, suggest that TGase C plays a role in myoblast attachment to the extracellular laminae during the early embryonal period.

Inactivation of alveolar macrophage transglutaminase by oxidants in cigarette smoke.

The present study demonstrates the existence of tissue transglutaminase in rabbit alveolar macrophages and measures the effects of cigarette smoke extracts on the activity of enzyme partially purified from these cells. The effects of smoke on transglutaminase purified from guinea pig liver are also measured. A water soluble component of gas‐phase cigarette smoke inhibits both enzymes in a dose‐dependent manner, with a maximum inhibition of 65% occurring at concentrations of smoke as low as 1% in the case of the rabbit enzyme. The chemical oxidant N‐chlorosuccinimide mimics the dose‐dependent inhibitory effects of cigarette smoke on both enzyme systems. The thiol reducing agent, dithiothreitol, can prevent enzyme inactivation mediated by both smoke and N‐chlorosuccinimide. Dithiothreitol is also capable of reversing inactivation mediated by cigarette smoke, but does not reverse inactivation caused by chemical oxidation with N‐chlorosuccinimide. These data suggest that cigarette smoke inhibits transglutaminase activity via an oxidative mechanism that may selectively attack the active‐site cysteine residue.

Protein kinase CK2 phosphorylates and interacts with deoxyhypusine synthase in HeLa cells

Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.

Expression of Transglutaminase C during the Prenatal Development of Human Submandibular Glands

The involvement of transglutaminase C (TGase C) in morphogenesis and cytodifferentiation during glandular tubule formation was addressed by immunolocalization of the protein at different stages of prenatal human submandibular gland development in 100 fetuses and 20 adult salivary glands. Immunocytochemical detection was carried out using a monospecific antibody to TGase C. The results showed TGase C reactivity in both acini and ducts early in development (from 10 to 14 weeks), followed by a marked increase in ductal activity and a decline in acinar activity up to 32 weeks. During the peak of reactivity at 25 to 32 weeks, staining was concentrated in the apical ends of the columnar cells. In the adult, staining was weakly and diffusely distributed in the striated and excretory ducts. Western blot analysis of the cellular extracts of pooled samples from various stages of salivary gland development showed a single strong band at 76 kDa early in development. This band became weaker after 32 weeks of prenatal development and in the adult. These findings of transient high expression of TGase C, which coincide with the development of tubulo-alveolar structure, suggest that TGase C may play a role in morphogenesis in human salivary gland development.

Immunohistochemical Evaluation of Transglutaminase C in Tumours of Salivary Glands

Transglutaminase C (TGase C), a family of Ca(2+)-dependent enzymes and an essential component in the cross-linking of peptide bonds, has been found to be a marker of epithelial differentiation with a possible role in cellular apoptosis, extracellular matrix stabilisation and Ca2+ binding, thereby having a potential role in tumour growth, differentiation and invasive behaviour. The expression of TGase C was evaluated in normal human salivary glands and their neoplastic lesions which included pleomorphic adenoma (n = 30), Warthins tumour (n = 5), adenoid cystic carcinoma (n = 10), acinic cell carcinoma (n = 5), mucoepidermoid carcinoma (n = 5) and control tissue specimens of normal oral mucosa and squamous cell carcinoma, using polyclonal antibody, the specificity of which was determined by Western blotting, generated by immunising rabbits with purified transglutaminase. The TGase C was observed in the epithelial cells in the control tissue specimens examined. Pleiomorphic adenoma revealed reaction products in luminal tumour cells, the non-luminal or modified myoepithelial cells and their plasmacytoid variants, squamous metaplastic cells and chondroid cells. Adenoid cystic carcinomas had tumour cells in the luminal cells of tubular and cribriform structures and the acinic cell carcinoma had from low to moderate immunoreactivity in the tumour cell component and a diffuse immunoreactivity in the stroma for TGase C. Mucoepidermoid carcinoma showed no reaction products in the mucous-producing cells, while intermediate and epidermoid cells had immunoreactivity in the cell cytoplasm. As the presence of TGase C in salivary gland tumours was confined to those tumour cells which form the predominant histomorphology in each tumour subtype, it may be suggested that these enzymes may have a potential role in the regulation of cellular function in neoplastic salivary tissues affecting tumour growth, differentiation and neoplastic behaviour.

Assessment of coagulation and fibrinolysis in synovial fluid of rheumatoid arthritis patients

Summary In order to understand the mechanism of fibrin deposition in the synovia during the progression of rheumatoid arthritis (RA), activities of coagulation factors, fibrinolytic enzymes, and corresponding inhibitors were measured in synovial fluid of 12 RA patients. Almost all of the synovial fluid (SF) fibrinogen molecules were found to be fragmented and some portions of the molecule that were slightly affected were found to be in a complex with albumin molecules. Immunohistochemical analysis showed deposits of insoluble fibrin in synovial membranes and pannus, in levels related to the progression of disease. Coagulation factor VII, VIII, X, XII, and XIII activities in SF were found to be 30 to 50% of plasma levels but factors II and V activities were less than 25% of plasma levels. Protein C and antithrombin III activities in SF were 30% of plasma levels. However, the two orders of magnitude, higher level of thrombin-antithrombin III (TAT) complex in SF than in plasma, suggests a steady activation of coagulation cascade in synovia. The observed increased levels of fibrinogen, TAT complexes, Bβ 15–42 peptide and plasminogen activator inhibitor-1 (PAI-1) in plasma are consistent with the systemic inflammatory state of RA patients. Although a 30% decrease in plasma levels of α2-plasmin inhibitor activity, and a 2-fold higher level of PAI-1 activity in SF were found, the presence of D-dimer and Bβ 15–42 peptide in SF suggests an active participation of plasmin in the fibrino(geno)lysis. In addition, elevated levels of elastase-α1-proteinase inhibitor complexes and of thrombin-increasable fibrinopeptide A in SF suggest the participation of leukocyte elastase in fibrin(ogen)olysis as well.