Regulatory peptides synthesis

The sizes of peptide molecules, i.e. the numbers of amino acid residues in their peptide chains, determine significant differences in the modes of (C) their transport through biological barriers and (D) their action on target cells. It will be shown below that the threshold for significant changes in the relevant properties of peptides is at four to five amino acid residues. Therefore, in the present book, at difference from the conventional classification, we will label peptides as short (short-chain peptides) only if they consist of two to four amino acids, i.e., if they are di-, tri, and tetrapeptides. All other peptides will be regarded as long (long-chain peptides).

The ribosomal synthesis of precursor proteins and their cleavage

The proteins that are synthesized in the cells of a macro-organism and in bacterial cells therein according to the Central Dogma of molecular biology, i.e., DNA→ mRNA → (ribosome + tRNAs + amino acids) → protein, are cleaved by enzymes at defined cleavage sites, i.e. peptide bonds within certain combinations (motifs) of amino acid residues, and in a definite order. This process, which is called limited proteolysis, consists of the following stages:

  1. The removal of the hydrophobic N-terminal signal peptide, which is responsible for transferring a protein molecule destined for export from a polyribosome to the surface of the rough endoplasmic reticulum.
  2. The proteolytic cleavage of bonds between some amino acid residues and the formation of peptides of different sizes.
  3. Posttranslational modifications of some amino acid residues of the resulting peptides (phosphorylation, acetylation etc.

The classic sites of enzymatic cleavage of a protein molecule are represented by combinations of two positively charged diaminomonocarboxylic acids, i.e. Lys-Arg (KR), Arg-Arg (RK) or Lys-Lys (KK). In different tissues, the proportions of cleavage at such sites vary from almost equal shares to a significant predominance of peptide bonds cleaved at the C-end side from Arg (Lys-Arg or Arg-Arg). Sometimes, cleavage takes place within the motif Arg-X-Lys/Arg-Arg (RXK/RR). Less commonly cleaved are peptide bonds involving only one basic amino acid, Arg or Lys [60]; however, such cases are important for the formation of short-chain regulatory peptides (see below). Cleavage sites may contain no arginine at all, e.g., neurotensin is cleaved in the brain not only at Arg-Arg (RR) and Pro-Arg (PR) bonds, but also at Pro-Tyr (PY) bonds.

The amino acid composition of endogenous regulatory peptides generally differs from that of proteins. We have compared [30] the amino acid composition of all proteins included in NIH Protein Database (, which is qualified as "a collection of sequences from several sources, including translations from annotated coding regions in GenBank, RefSeq and Third Party Annotation, as well as records from SwissProt, PIR, PRF, and PDB", with that of more than 150 human regulatory peptides reviewed earlier (Table 1). For this, we combined all amino acid sequences available into one "word" comprised of 11 billion letters and calculated the percent contents of each letter (e.g. A for alanine, C for cysteine etc.) The same was done with amino acid sequences of 155 regulatory peptides.
Khavinson peptides
The validity of the above comparison is limited by three considerations:

  1. Two samples of very discordant sizes are compared: the sum of amino acid residues in the proteins is 106 times greater than that in the regulatory peptides.
  2. Data on all proteins from different species are compared with data on only human regulatory peptides.
  3. In databases used, some important classes of proteins are underrepresented, e.g., membrane proteins, which are more difficult for isolation than cytoplasmic proteins are .

With all the above caveats, the following may be noted:

  1. Compared with the proteins, the regulatory peptides (which are long-chain in the present case) contain more of the amino acids G, S, R, K, P, F, Q, Y, H, M, W, and C and less of L, A, V, E, T, I, D, and N.
  2. The amino acids that are the most prevalent in the proteins are the least prevalent in the regulatory peptides, and vice versa.

The greatest is the difference (1:2) in cysteine (Cys, C), which is required for disulfide (S-S) bond formation needed to stabilize polypeptide conformation. The reason of the relatively high presence of glycine (Gly, G) in peptides is probably that due to its simple structure it does not hamper conformational changes and thus facilitates the formation of appropriate spatial configurations. On a whole, it may be conclude that the bulk of the most prevalent amino acids in proteins relate to their functionally inactive domains.

The short-chain regulatory peptides designed at Saint-Petersburg Institute of Bioregulation and Gerontology (Khavinson Peptides®), comprise only seven of all proteinogenic amino acids, and the comparative rates of their presence in the peptides is as follows: E > D > G = K = W > A = R. The most prevalent, according to this rating, are the negatively charged (at pH = 7) glutamic acid (Glu, E) and aspartic acid (Asp, D).

The non-ribosomal synthesis of peptides

Peptide bonding of amino acids outside of ribosomes results in the de novo formation of regulatory peptides, including short-chain ones, such as carnosine (Ala-His, AH) and glutathione (Glu-Cys-Gly, ECG) (see [36, 39]).

Fritz Lipmann, a Nobel Laureate in physiology and medicine (1953), has shown that the non-ribosomal enzymatic synthesis is common in microorganisms that produce peptide antibiotics [40]; therefore, peptides of the non-ribosomal origin found in animals, including humans, may be produced by microorganisms rather than by body cells. This phenomenon has been studied mainly as represented by antimicrobial peptides derived from bacteria entering the gastrointestinal tract with foods, such as milk or honey [9, 10]. Some amounts of peptides of the non-ribosomal origin may be found in foodstuffs themselves, and certain short-chain peptides of this sort may enter the inner milieu of human body by crossing the intestinal barrier (see below).

A characteristic feature of peptides of the non-ribosomal origin is that their proteinogenic amino acids may be glycosylated, acylated, halogenated, or hydroxylated; they also may carry N-methyl or N-formyl groups. Besides that, such peptides often contain unusual or non-proteinogenic amino acids (see [36]) and D-amino acids.

The molecules of such peptides often comprise cyclic structures. For example, the soil-dwelling fungi Tolypocladium inflatum synthesize, in a non-ribosomal manner, a cyclic undecapeptide cyclosporine [7]. The primary structure of cyclosporine A [27] may be represented, in a simplified way, as the following:

cyclo (Leu-Val-Thr-Abu-Sar-Leu-Val-Leu-Ala-ala-Leu)
Here, the non-proteinogenic amino acids are shown with bold abbreviations:
Abu = α-aminobutyric acid (non-proteinogenic amino acid);
Sar = sarcosine (an intermediate and byproduct in glycine synthesis);
and ala = D-Ala = D-alanine.

The non-ribosomal synthesis can result not only in short-chain peptides, such as carnosine and glutathione, but in microorganisms, in peptides having up to several scores of amino acid residues, as reviewed in several publications.

It was repeatedly hypothesized that the non-ribosomal synthesis is an ancient pathway of peptide production, which in the course of evolution was replaced by a more progressive ribosomal synthesis. Probably, this is true. However, the question remains when and how nonribosomal peptide synthetases (NRPSs), which themselves are proteins produced at ribosomes and indispensable for non-ribosomal peptide synthesis, could emerge.

As to regulatory peptides in the brain, glutathione, a product of the non-ribosomal protein synthesis, is found associated with the synaptic membranes in mammalian brain and is suggested to be a candidate neurotransmitter. However, Michel Chrétien and coauthors from Université de Montréal have put forward, as long ago as in the 1970s, the suggestion, which later became generally accepted, that all neuropeptides are synthesized as precursor protein molecules, which are then fragmented by enzymes.