Humanized amino acid sequences

Based on the above principles, it might be assumed that a therapeutic protein obtained by direct extraction from human sources (e.g. some antibody preparations) or produced via recombinant expression of a human gene/cDNA sequence (e.g. recombinant human hormones or cytokines) would be non-immunogenic in humans whereas foreign therapeutic proteins (e.g. non-engineered monoclonal antibodies) would stimulate a human immune response. This general principle holds in many cases, but not all. So why do therapeutic proteins of human amino acid sequences have the potential to trigger an immune response Potential reasons can include ... 

Figure 8.4. Amino acid sequence of porcine insulin is depicted in (a). Trypsin cleavage sites are also indicated. Trypsin therefore effectively removes the insulin carboxy-terminus B chain octapeptide. The amino acid sequence of human insulin differs from that of porcine insulin by only one amino acid residue. Porcine insulin contains an alanine residue at position 30 of the B-chain, whereas human insulin contains a threonine residue at that position. Insulin exhibiting a human amino acid sequence may thus be synthesized from porcine insulin by treating the latter with tr5q)sin, removal of the C terminus fragments, generated and replacement of this with the synthetic octapeptide shown in (b). Reproduced by permission of John Wiley Sons Ltd from Walsh Headon (1994)...

Biosynthetic Human Insulin from E. coli. Insulin [9004-10-8] a polypeptide hormone, stimulates anaboHc reactions for carbohydrates, proteins, and fats thereby producing a lowered blood glucose level. Porcine insulin [12584-58-6] and bovine insulin [11070-73-8] were used to treat diabetes prior to the availabiHty of human insulin [11061 -68-0]. AH three insulins are similar in amino acid sequence. EH LiHy s human insulin was approved for testing in humans in 1980 by the U.S. EDA and was placed on the market by 1982 (11,12). 

The amino acid sequences of hCS-A, hCS-B, and hCS-V are shown in relation to GH in Figure 1. The sequence of hCS-V is predicted from the DNA coding sequence and apparentiy does not possess amino acids 8—55 relative to GH and the other hCS molecules. It is not certain whether hCS-V is expressed or what function it may have. Human CS-A and hCS-B share approximately 85% identity with GH and also possess the disulfide bonds between Cys 53—165 and Cys 182—189 which produce the long and short S—loops characteristic of the PRL/GH family. 

Fig. 1. Amino acid sequence for the A- and B-chains of human iasulin [11061-68-0] where soHd lines denote disulfide bonds. Porciae iasulin [12584-58-6] differs by one amino acid ia the B-chaia where alanine replaces threonine at positioa 30. Boviae iasulia [11070-73-8] differs by three amino acids. la the A-chain alanine replaces the threonine at positioa 8 and valine replaces the isoleuciae at position 10. In the B-chain there is an alanine at position 30.

Eig. 6. Amino acid sequence of human (H) and porcine (P) calcitonins. 

LDH-x is one of the best characterized antigens and its amino acid sequence is known. A synthetic peptide based on a portion of the molecule has been shown to reduce fertUity in laboratory animals. The nucleotide sequence coding for human LDH-x has been defined and engineered into an expression vector system (121). 

Figure S.S Amino acid sequence of p strands 2 3 4 in human plasma retinol-binding protein. The sequences are listed in such a way that residues which point into the barrel are aligned. These hydrophobic residues are arrowed and colored green. The remaining residues are exposed to the solvent.

Given a large population of individuals, a considerable number of sequence variants can be found for a protein. These variants are a consequence of mutations in a gene (base substitutions in DNA) that have arisen naturally within the population. Gene mutations lead to mutant forms of the protein in which the amino acid sequence is altered at one or more positions. Many of these mutant forms are neutral in that the functional properties of the protein are unaffected by the amino acid substitution. Others may be nonfunctional (if loss of function is not lethal to the individual), and still others may display a range of aberrations between these two extremes. The severity of the effects on function depends on the nature of the amino acid substitution and its role in the protein. These conclusions are exemplified by the more than 300 human... 

Apelins and the Apelin Receptor. Figure 2 Sequence alignment of mammalian and amphibian apelin-36 amino-acid sequences, indicates residues conserved across all the species shown. Residues which differfrom the human sequence are highlighted in red. 

NOS (eNOS, NOS IH, NOS3). Classically, nNOS and eNOS were considered constitutive enzymes, whereas iNOS is cytokine-induced. Recent evidence suggests that nNOS and eNOS are also subject to important regulation of expression [1 ]. Within the human species, amino acid sequences of the three NOS isoforms share 52-58% identity. Each isoform is well conserved across mammalian species (>90% amino acid identity for nNOS and eNOS, >80% for iNOS). NOS enzymes exist in organisms as low as nematodes, protozoa, and even in plants (Fig. 1). 

About 78 human genes encoding a variety of K+ channels and auxiliary subunits have been identified (Fig. 1 Table 1). While the K+ channels are diverse, they share with a unique conducting pore highly selective for K+ ions. TheK+ channels ar e tetramers composed of four a subunits that form the conducting pore. On the basis of primary amino acid sequence of a subunit, K+ channels can be classified into three major families (Fig. 2). 

The common C-terminal amino acid sequence required for exerting activity at tachykinin receptors is shown in bold endokinin C and D lack the C-terminal Met and are almost devoid of affinity at these receptors. In red, the sequence of neurokinin A of which neuropeptide-gamma and neuropeptide-kappa are elongated forms and neurokinin A (3-10) is a product of beta or gamma-TAC1 mRNAs or an NKA metabolite active at tachykinin receptors. In blue, the sequence of human HK-1 of which endokinin A and B are elongated forms. 

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