3 research outputs found
Site-Specific Derivatization of Avidin Using Microbial Transglutaminase
Avidin conjugates have several important
applications in biotechnology
and medicine. In this work, we investigated the possibility to produce
site-specific derivatives of avidin using microbial transglutaminase
(TGase). TGase allows the modification of proteins at the level of
Gln or Lys residues using as substrate an alkyl-amine or a Gln-mimicking
moiety, respectively. The reaction is site-specific, since Gln and
Lys derivatization occurs preferentially at residues embedded in flexible
regions of protein substrates. An analysis of the X-ray structure
of avidin allowed us to predict Gln126 and Lys127 as potential sites
of TGase’s attack, because these residues are located in the
flexible/unfolded C-terminal region of the protein. Surprisingly,
incubation of avidin with TGase in the presence of alkylamine containing
substrates (dansylcadaverine, 5-hydroxytryptamine) revealed a very
low level of derivatization of the Gln126 residue. Analysis of the
TGase reaction on synthetic peptide analogues of the C-terminal portion
of avidin indicated that the lack of reactivity of Gln126 was likely
due to the fact that this residue is proximal to negatively charged
carboxylate groups, thus hampering the interaction of the substrate
at the negatively charged active site of TGase. On the other hand,
incubation of avidin with TGase in the presence of carbobenzoxy-l-glutaminyl-glycine in order to derivatize Lys residue(s) resulted
in a clean and high yield production of an avidin derivative, retaining
the biotin binding properties and the quaternary structure of the
native protein. Proteolytic digestion of the modified protein, followed
by mass spectrometry, allowed us to identify Lys127 as the major site
of reaction, together with a minor modification of Lys58. By using
TGase, avidin was also conjugated via a Lys-Gln isopeptide bond to
a protein containing a single reactive Gln residue, namely, Gln126
of granulocyte-macrophage colony-stimulating factor. TGase can thus
be exploited for the site-specific derivatization of avidin with small
molecules or proteins
Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase
The transglutaminase (TGase) from <i>Streptomyces
mobaraensis</i> catalyzes transamidation reactions in a protein
substrate leading
to the modification of the side chains of Gln and Lys residues according
to the A-CONH<sub>2</sub> + H<sub>2</sub>N-B → A-CONH-B + NH<sub>3</sub> reaction, where both A and B can be a protein or a ligand.
A noteworthy property of TGase is its susbstrate specificity, so that
often only a few specific Gln or Lys residues can be modified in a
globular protein. The molecular features of a globular protein dictating
the site-specific reactions mediated by TGase are yet poorly understood.
Here, we have analyzed the reactivity toward TGase of apomyoglobin
(apoMb), α-lactalbumin (α-LA), and fragment 205–316
of thermolysin. These proteins are models of protein structure and
folding that have been studied previously using the limited proteolysis
technique to unravel regions of local unfolding in their amino acid
sequences. The three proteins were modified by TGase at the level
of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing
several Gln and Lys residues, the sites of TGase derivatization occur
over restricted chain regions of the protein substrates. In particular,
the TGase-mediated modifications occur in the “helix F”
region in apoMb, in the β-domain in apo-α-LA in its molten
globule state, and in the N-terminal region in fragment 205–316
of thermolysin. Interestingly, the sites of limited proteolysis are
located in the same chain regions of these proteins, thus providing
a clear-cut demonstration that chain flexibility or local unfolding
overwhelmingly dictates the site-specific modification by both TGase
and a protease
Structural and Antimicrobial Features of Peptides Related to Myticin C, a Special Defense Molecule from the Mediterranean Mussel Mytilus galloprovincialis
Mussels (Mytilus spp.) have a large
repertoire of cysteine-stabilized α,β peptides, and myticin
C (MytC) was identified in some hundreds of transcript variants after
in vivo immunostimulation. Using a sequence expressed in Italian mussels,
we computed the MytC structure and synthesized the mature MytC and
related peptide fragments (some of them also prepared in oxidized
form) to accurately assess their antibacterial and antifungal activity.
Only when tested at pH 5 was the reduced MytC as well as reduced and
oxidized fragments including structural β-elements able to inhibit
Gram-positive and -negative bacteria (MIC ranges of 4–32 and
8–32 μM, respectively). Such fragments caused selective Escherichia coli killing (MBC of 8–32 μM)
but scarcely inhibited two fungal strains. In detail, the antimicrobial
β-hairpin MytC[19–40]S<sub>OX</sub> caused membrane-disrupting
effects in <i>E. coli</i> despite its partially ordered
conformation in membrane-mimetic environments. In perspective, MytC-derived
peptides could be employed to protect acidic mucosal tissues, in cosmetic
and food products, and, possibly, as adjuvants in aquaculture