Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta)

Abstract

In this work, transcripts encoding three metallothioneins from Ulva compressa (UcMTs) were amplified: The 5′and 3′ UTRs by RACE-PCR, and the open reading frames (ORFs) by PCR. Transcripts encoding UcMT1.1 (Crassostrea-like), UcMT2 (Mytilus-like), and UcMT3 (Dreissena-like) showed a 5′UTR of 61, 71, and 65 nucleotides and a 3′UTR of 418, 235, and 193 nucleotides, respectively. UcMT1.1 ORF encodes a protein of 81 amino acids (MW 8.2 KDa) with 25 cysteines (29.4%), arranged as three motifs CC and nine motifs CXC; UcMT2 ORF encode a protein of 90 amino acids (9.05 kDa) with 27 cysteines (30%), arranged as three motifs CC, nine motifs CXC, and one motif CXXC; UcMT3 encode a protein of 139 amino acids (13.4 kDa) with 34 cysteines (24%), arranged as seven motifs CC and seven motifs CXC. UcMT1 and UcMT2 were more similar among each other, showing 60% similarity in amino acids; UcMT3 showed only 31% similarity with UcMT1 and UcMT2. In addition, UcMTs displayed structural similarity with MTs of marine invertebrates MTs and the terrestrial invertebrate Caenorhabtidis elegans MTs, but not with MTs from red or brown macroalgae. The ORFs fused with GST were expressed in bacteria allowing copper accumulation, mainly in MT1 and MT2, and zinc, in the case of the three MTs. Thus, the three MTs allowed copper and zinc accumulation in vivo. UcMTs may play a role in copper and zinc accumulation in U. compressa.

Copper-induced concomitant increases in photosynthesis, respiration, and C, N and S assimilation revealed by transcriptomic analyses in Ulva compressa (Chlorophyta)

“…The levels of transcripts encoding MTs, antioxidant enzymes, and enzymes involved in ASC and GSH synthesis, were increased in the alga exposed to copper excess [16]. Finally, transcripts encoding three MTs, MT1, MT2 and MT3, were cloned from U. compressa and their overexpression in bacteria allowed the accumulation of copper and zinc in vivo [17]. Thus, U. compressa may accumulate copper in its tissue through the binding of copper ions to GSH, PCs and MTs and this alga could represent a useful tool for phycoremediation of seawater contaminated with heavy metals.…”

Mechanisms of Copper Tolerance, Accumulation, and Detoxification in the Marine Macroalga Ulva compressa (Chlorophyta): 20 Years of Research

“…The overexpression of Fucus MT in Escherichia coli allowed the accumulation of arsenite and arsenate, but not cadmium, zinc, or lead [34]. It was recently shown that three MTs of the green alga U. compressa allowed the accumulation of copper and zinc in E. coli [25]. Thus, the excess of copper and other heavy metals induced the synthesis of PCs and MTs in marine macroalgae allowing heavy metal accumulation.…”

“…Surprisingly, the UcMTs' amino acid sequences were not closely related with MTs from red or brown marine macroalgae. The open reading frames (ORFs) of UcMTs were cloned in an expression vector that allow the synthesis of a fusion protein containing in N-terminal position a GST of the flatworm Shistosoma japonicum followed by UcMT [25]. Importantly, S. japonicum's GST contains a single cysteine in its amino acid sequence and, thus, is unable to bind heavy metal ions.…”

“…Importantly, S. japonicum's GST contains a single cysteine in its amino acid sequence and, thus, is unable to bind heavy metal ions. GST-UcMTs were overexpressed in E. coli and their overexpression allowed the accumulation of proteins having the predicted sizes and GST tail that was detected using a specific antibody [25]. More importantly, UcMTs overexpressed in E. coli allowed the accumulation of copper and zinc in vivo; UcMT1 and UcMT2 allowed a higher accumulation of copper compared with UcMT3 and control bacteria, and the three UcMTs allowed similar accumulation of zinc levels [25].…”

“…In marine brown and red macroalgae, there is only one MT, and until recently the only cloned and expressed MT was the brown macroalga Fucus vesiculosus MT [ 23 , 24 ]. Recently, three MTs were identified in the green macroalga Ulva compressa [ 25 ].…”