Functional characterization of naturally occurring transglutaminase 2 mutants implicated in early-onset type 2 diabetes

Abstract

Transglutaminase 2 (TG2) is an enzyme with diverse biological functions. TG2 catalyzes transamidation reactions, has intrinsic kinase activity, and acts as a G-protein in intracellular signaling. TG2 (Tgm2)-null mice are glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Furthermore, three naturally occurring missense mutations in the human TGM2 gene, corresponding to amino acid substitutions of Met330Arg, Ile331Asn, and Asn333Ser in the TG2 protein, have been reported and found to be associated with early-onset type 2 diabetes. However, their effect on TG2 function is not fully understood. To determine this, we have reproduced naturally occurring mutations in TG2 using site-directed mutagenesis. Overexpression of Myc-TG2 mutants in INS-1E cells resulted in a reduction of GSIS in comparison with cells overexpressing wild-type Myc-TG2 (WT-TG2). The maximum reduction was found in cells overexpressing Ile331Asn-TG2 (32%) followed by Met330Arg-TG2 (20%), and the least in Asn333Ser-TG2 (7%). Enzymatic analysis revealed that TG2 mutants have impaired transamidation and kinase activities in comparison with WT-TG2. GTP-binding assays showed that TG2 mutants also have altered GTP-binding ability, which is found to be modulated in response to glucose stimulation. Collectively, these data suggest that naturally occurring mutations in TG2 affect transamidation, kinase, and GTP-binding functions of TG2. While reduced insulin secretion, as a result of naturally occurring mutations in TG2, is due to the impairment of more than one biological function of TG2, it is the transamidation function that appears to be impaired during the first phase, whereas the GTP-binding function affects the second phase of insulin secretion.

Glucose Homeostasis in Mice Is Transglutaminase 2 Independent

“…Our data, however, are contrary to prior reports implicating a role for TG2 in insulin secretion [3], [18]–[21]. One of these studies [3] used mixed-strain TG2 −/− mice generated independently by another group [5].…”

“…Thus, it is possible that unexpected contributions from the 129 genome that interact deleteriously with the B6 genome might explain the glucose tolerance and impaired insulin secretion reported by Bernassola et al ., (2002) using their mixed-strain TG2 −/− mice. Another study [21] recently reported impaired insulin secretion by islets isolated from that same TG2 −/− mouse line, now reported to be on a B6 background, although the number of generations of backcrossing and, thus, the genomic homogeneity of the line was not stated. Other studies implicating transamidase activity in insulin secretion [18]–[20] have used small molecule inhibitors such as methylamine, cystamine and dansylcadaverine, which, in addition to being alternate substrates that competitively inhibit transglutaminases [36], are well-known to have many biological effects, including antioxidant activity, inhibition of a range of cysteine proteases, as well as inhibition of endocytosis [37]–[39].…”

“…The transamidase activity of TG2 has been suggested to modulate the exocytosis step of glucose-stimulated insulin secretion (GSIS) that accompanies calcium influx into pancreatic β cells [18]–[20]. Consistent with TG2 involvement in insulin release, pancreatic islets from TG2 null mice [5] have been reported to secrete less insulin, relative to wild-type (WT) islets, in response to a high glucose challenge [3], [21]. This was reflected in lower blood insulin and higher blood glucose levels in TG2 −/− mice following intraperitoneal glucose loading [3].…”

Diadenylate cyclase evaluation of ssDacA (SSU98_1483) in Streptococcus suis serotype 2

“…To analyze the ATP binding to ssDacA , ssDacA DGA , and ssDacA RHR , an ATP-Sepharose binding assay was used in a binding procedure previously described with minor modifications (Dias et al, 2009;Reese and Boothroyd, 2011;Bai et al, 2012;Salter et al, 2012). Briefly, purified recombinant proteins and bovine serum albumin, used as negative control, were incubated at 4°C for 1 h with a 50-mL bed volume of g-linked ATP-Sepharose (Innova Biosciences, Cambridge, UK) in 50 mM Tris, pH 7.5, and 150 mM NaCl, respectively.…”

Insights into Transglutaminase 2 Function Gained from Genetically Modified Animal Models

“…TG2 has been reported to be a candidate gene for MODY, with heterozygous mutations that impair transamidase activity (M330R, I331N, or N333S) having been identified in one or more affected individuals from three different families (Bernassola et al 2002b;Porzio et al 2007). In vivo (Bernassola et al 2002b) and less convincing in vitro (Salter et al 2012) studies have reported impaired glucose-stimulated insulin secretion by pancreatic islets from TG2 À/À mice [(De Laurenzi and Melino 2001), mixed-strain 129-B6 or B6, respectively] relative to wild-type mice. However, another study (performed according to the recommended guidelines for animal husbandry and study design), using congenic B6 and 129 TG2 À/À lines [ (Nanda et al 2001) backcrossed 12 generations to the respective inbred strains], showed no impairment in vivo or in vitro of glucose-stimulated insulin secretion by pancreatic islets from TG2 À/À mice relative to wild-type littermates .…”