Karin Hjalmarsson

The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide.

The nucleotide sequence of a 4.6‐kb SalI‐EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K‐12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21‐K polypeptide (unknown function), tRNA‐(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6‐kb DNA fragment encodes a 48‐K and a 16‐K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2‐terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co‐transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA‐(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non‐coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA‐(m1G)methyltransferase molecules found in the cell.

Optimising the signal peptide for glycosyl phosphatidylinositol modification of human acetylcholinesterase using mutational analysis and peptide-quantitative structure–activity relationships

Glycosyl phosphatidylinositol (GPI)-modified proteins have a C-terminal signal peptide (GPIsp) that mediates the addition of a GPI-anchor to an amino acid residue at the cleavage and modification site (omega-site). Within the GPIsp, a stretch of hydrophilic amino acid residues are found which constitutes the spacer region that separates the omega-site residue from a hydrophobic C-terminus. Deletions and insertions into the spacer region of human acetylcholinesterase (AChE) show that the length of this spacer region is very important for efficient GPI-modification. Surprisingly, the natural length of the spacer region in human AChE was not optimal for the highest degree of GPI modification. The importance of the two adjacent residues downstream of the omega-site, the omega+1 and omega+2 residues, was investigated by peptide-quantitative structure-activity relationships (Peptide-QSAR). A model was made that predicts the efficiency of the GPI modification when these residues are substituted with others, and suggests important features for these residues. The most preferred omega+1 and omega+2 residues, predicted by the model, in combination with an ideal spacer length resulted in an optimised GPIsp. This mutant protein is more efficiently GPI-modified than any mutant AChE tested thus far.

Cloning and sequencing of a cDNA encoding human milk β-casein

A cDNA of 1065 bp encoding the human milk β‐casein was cloned and sequenced using a synthetic oligodeoxyribonucleotide probe and a human mammary gland library. The nucleotide (nt) sequence contained an open reading frame sufficient to encode the entire amino‐acid (aa) sequence of a β‐casein precursor protein consisting of 210 aa and a signal peptide of 15 aa. The nt sequence shows 45–62% homology to those of bovine, ovine, rat, and mouse β‐caseins. The highly phosphorylated site, which is responsible for the calcium‐binding capacity of β‐casein, the signal peptide, and a sequence encoding for an inhibitor to the angiotensin‐converting enzyme seem highly conserved among the β‐caseins with known sequences.

Residues in Torpedo californica acetylcholinesterase necessary for processing to a glycosyl phosphatidylinositol-anchored form

Acetylcholinesterase from Torpedo californica (TcAChE) can be found as a glycosyl phosphatidylinositol (GPI)-anchored, membrane associated form. The C-terminal amino-acid sequence of the precursor protein resembles the signal peptide sequence found in proteins and enzymes destined for GPI-modification. Characteristics of such a signal peptide are a relatively polar stretch of amino acids, separating a cleavage- and modification-site (omega-site) residue from a hydrophobic C-terminus. We have introduced mutations, both at putative omega-sites and in the hydrophobic region, and analysed their effects on GPI-anchoring of TcAChE. Our results show that substitution of all three Ser residues in the region Ser542-Ser544 prevents GPI-modification and membrane anchoring. Individual substitution of each of these residues resulted in no or only a minor effect on the modification. We therefore conclude that more than one residue within this sequence can be utilised as the omega-site. Our analyses of double substitutions indicated that Ser543 and Ser544 are the preferred residues for GPI-modification. Moreover, the hydrophobic region is shown to be essential for GPI-anchoring of TcAChE.

Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli.

During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.

Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase

The three-dimensional crystal structure of the glycosyl phosphatidylinositol (GPI)-modified form of Torpedo acetylcholinesterase reveals the participation of Arg-44 and Glu-92 in a salt bridge and a hydrogen bond between Asp-93 and Tyr-96. To investigate the biological significance of these interactions, we have made amino acid replacements in this form of AChE: R44E, R44K, E92Q, E92L, D93N, and D93V. None of the introduced mutations affected the production of the acetylcholinesterase polypeptide significantly. However, the mutations introduced at position 92, as well as the D93V and R44E mutations, resulted in a total loss of surface located, active acetylcholinesterase. Replacement of Asp-93 with Asn resulted in a reduced amount of active enzyme. This mutant enzyme was indistinguishable from the wild-type enzyme regarding catalytic constants, but was more sensitive to thermal inactivation. The results show that the salt bridge and hydrogen bond involving residues Arg-44, Glu-92, and Asp-93 have important structural roles and are needed for correct folding, required for transport to the cell surface of TcAChE. The GPI-modified form of acetylcholinesterase is a disulfide bonded dimer. Cys-537 is shown to be required for the formation of the intersubunit disulfide bond in the dimer. Replacement with Ser resulted in the production of an enzyme, that migrates as a monomer upon non-reducing SDS-PAGE and has a lower stability compared to the dimeric wild-type enzyme.

Structurally Important Residues in the Region Ser91 to Asn98 of Torpedo Acetylcholinesterase

In cholinesterases and cholinesterase-like proteins the region Ser91 to Asn98, numbering according to acetylcholinesterase (AChE) from T. californica, shows a high degree of conservation. In this region two negatively charged amino acids, Gluy and Asp93, are found as well as a cysteine, Cys94, which is disulphide bonded to Cys67 1. The loop created by the disulphide bons contains Trp84, which has been identified as part of the anionic subsite of the active site2. This suggests that the highly conserved region could have an important structural role. To gain insight into the possible role of G1u92 and Asp93 in maintaining the structure and function of AChE, we have made amino acid replacements by site-directed mutagenesis.

Optimization of the Signal Peptide for Glycosyl Phosphatidylinositol-(GPI)-Modification of Human Acetylcholinesterase

Acetylcholinesterase (AChE, EC 3.1.1.7.) is an enzyme generally known to hydrolyze the neurotransmitter acetylcholine, in neuromuscular junctions. Yet, other enzyme forms of AChE are found, such as the glycosyl phosphatidylinositol (GPI)-anchored form. This enzyme form is predominantly found attached to the plasma membrane of the red blood cells, by a GPI moiety. The biological role of this enzyme form is unclear but it has been suggested to control proliferation and differentiation of pluripotent stem cells from the bone marrow1,2.

Residues in the C-Terminus of Torpedo californica Acetylcholinesterase Important for Modification into a Glycophospholipid Anchored Form

Acetylcholinesterase (AChE) is an enzyme that exists in several structurally distinct forms. Two major forms of AChE molecules are found in the electric organ of Torpedo californica. A hydrophilic form that is attached by a collagen-like tail to the basal lamina in the synaptic cleft, and a hydrophobic dimeric form (G2-AChE) that is attached to the cell membrane via a glycosyl-phosphatidyl inositol (GPI) anchor. These two different forms arise due to alternative splicing of two exons, exon 5 and exon 6. Exon 5 encodes the last 31 carboxy-terminal amino acids found in the hydrophobic form of AChE. This C-terminal peptide (GPIsp) contains the signal for GPI modification. The GPI-moiety is attached to a specific amino acid residue, encoded by exon 5, at the cleavage/modification site or co-site. This residue is found about 20 amino acids upstream of the C-terminus. Studies of other GPI-modified proteins have not revealed a definitive consensus amino acid sequence in the C-terminal region, but some characteristics are found. The first 2 amino acids downstream of the co-site, positions ω+1 and ω+2, are believed to interact with the active site of a putative transamidase, catalyzing the GPI-modification reaction. The ω+2 position in the GPIsp is the most conserved residue. In natural proteins only a few amino acids (Gly, Ala, Ser and Thr) are found in this position (Kodukula et al., 1993). These residues are followed by a stretch of 5 to 10 small and relatively polar amino acids, the “spacer region”. The “spacer region” is followed by a stretch of 10–15 hydrophobic amino acids.