The complete amino acid sequence of human brain-derived acidic fibroblast growth factor

Acidic fibroblast growth factor is a potent mitogen for a variety of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of human brain-derived acidic fibroblast growth factor has been determined from amino terminal sequence analysis and carboxypeptidase A digestion of the whole protein and sequence analyses of peptides generated by tryptic, Staphylococcus aureus V8 protease and cyanogen bromide cleavages. A potential Asn-Gly-Ser glycosylation sequence is present in the human protein. The complete amino acid sequence is compared to that of the equivalent protein purified from bovine brain.     

The complete amino acid sequence of the shorter form of human basic fibroblast growth factor receptor deduced from its cDNA

We have isolated a full-length cDNA for human basic fibroblast growth factor (bFGF) receptor-like protein from a human placenta cDNA library. Determination of the nucleotide sequence of the cDNA allows elucidation of the complete amino acid sequence of the receptor (731 amino acids) which has two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor has remarkable amino acid similarity (98% identity) to the shorter form of murine bFGF receptor reported recently (H.H.Reid et al. (1990) Proc.Natl.Acad.Sci. USA 87, 1596-1600). The receptor described here is expected to be the shorter form of human bFGF receptor.     

Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells.     

Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.     

The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin

The complete amino acid sequence of human insulin-like growth factor I (IGF-I), a polypeptide isolated from serum, has been determined. IGF-I is a single chain polypeptide of 70 amino acid residues cross-linked by three disulfide bridges. The calculated molecular weight is 7649. IGF-I displays obvious homology to proinsulin: positions 1 to 29 are homologous to insulin B chain and positions 42 to 62 to insulin A chain. A shortened "connecting" peptide with 12 residues (positions 30 to 41) compared to 30 to 35 in proinsulins shows no homology to proinsulin C peptide. An octapeptide sequence at the COOH-terminal end is also a feature not found in proinsulins. The number of differences in amino acid positions between IGF-I and insulins suggests that duplication of the gene of the common ancestor of proinsulin and IGF occurred before the time of appearance of the vertebrates. Of the 19 residues known to be invariant in all insulins so far sequenced, only glutamine A5 and asparagine A21 are replaced in IGF-I by glutamic acid and alanine, respectively. The fact that all half-cystine and glycine residues and most nonpolar core residues of the insulin monomer are conserved is compatible with a three-dimensional structure of IGF-I similar to that of insulin.     

Sterilization of epidermal growth factor with supercritical carbon dioxide and peracetic acid; analysis of changes at the amino acid and protein level

Aseptic processing and terminal sterilization become increasingly challenging as medical devices become more complex and include active biologics. Terminal sterilization is preferred for patient safety and production costs. We aimed to determine how sterilization using supercritical CO₂ (scCO₂) with low levels of peracetic acid (PAA) affects amino acids and human epidermal growth factor (EGF) as a model protein. In a benchtop reactivity test, the amino acids methionine, tryptophan, arginine and lysine reacted with low levels of PAA in solution. At PAA levels used for scCO₂ sterilization, however, mass spectrometry only identified oxidative adducts on methionine and tryptophan. Mass spectrometry analysis of EGF exposed to scCO₂/PAA identified oxidative adducts on residues Met₂₁, Trp₄₉ and Trp₅₀, as well as a low level of truncations after residues Trp₄₉ and Trp₅₀. Importantly, processing of EGF in solution with scCO₂ did not affect its native conformation, and sterilized EGF maintained its activity in cell proliferation assays. When processing samples in lyophilized form with scCO₂/PAA, amino acids did not react with PAA and the presence of adducts was strongly reduced on methionine and tryptophan, both as single amino acids and in EGF. Truncation after tryptophan residues did not occur. EGF sterilized in the lyophilized form retained its activity when processing occurred with added moisture. These results have significant implications for the maintenance of biological function in sterilized decellularized scaffolds and the ability to manufacture terminally sterilized combination devices containing therapeutic peptides or proteins.     

N-terminal amino acid sequence of an insect neurohormone, melanization and reddish coloration hormone (MRCH): heterogeneity and sequence homology with human insulin-like growth factor II

Catecholamine release from chromaffin cells in response to carbamylcholine and high K+ is transient. Monitoring intracellular free calcium ([Ca2+]i) using quin2 demonstrated a transient rise in [Ca2+]i in response to carbamylcholine. The termination of secretion due to carbamylcholine is probably a consequence of the return of [Ca2+]i to resting levels as the nicotinic receptors desensitise. Depolarisation with 55 mM K+ led to a long-lasting rise in [Ca2+]i which persisted after the secretory response had terminated. The maintained rise in [Ca2+]i appeared to be due to continued opening of verapamil-sensitive Ca2+ channels. These results suggest that inactivation of voltage-dependent Ca2+ channels does not account for the transient nature of the secretory response in chromaffin cells.     

Solution synthesis and biological activity of human pleiotrophin, a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five disulfide bonds.

Human pleiotrophin (hPTN), a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five intramolecular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility of our strategy using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermediates, Boc-(1-64)-OH and H-(65-136)-OBzl, in CHL/TFE (3:1; v/v) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). After removal of all protecting groups using the HF procedure followed by treatment with Hg(OAc)2, the fully deprotected peptide was subjected to an oxidative folding reaction. The product was confirmed as having the correct disulfide structure by examining the cystine peptides obtained by enzymatic digestions, and as possessing the same biological activities as those of the natural product. The N- and C-terminal half domains (1-64 and 65-136) were also synthesized, and measurement of their biological activities indicated that the C-terminal half domain displays almost all the activities of the full-length molecule, whereas the N-terminal half domain shows almost no activity. From these results, we were able to confirm that the C-terminal half domain is responsible for the expression of biological activities in the same manner as human midkine (hMK), another heparin-binding neurotrophic growth factor.     

Isomers of epidermal growth factor with Ser --> Cys mutation at the N-terminal sequence: isomerization, stability, unfolding, refolding, and structure

The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys(6)-Cys(20), Cys(14)-Cys(31), and Cys(33)-Cys(42). The connection of Cys(6) and Cys(20) forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738-1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845-4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser --> Cys mutation at Ser residues (Ser(2), Ser(4), and Ser(9)) flanking Cys(6). This allows competition between Cys(6) and mutated Cys(2), Cys(4), and Cys(9) to link with Cys(20) and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys(6)-Cys(20) is favored over Cys(2)-Cys(20) and Cys(4)-Cys(20) by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys(9)-Cys(20) is thermodynamically more stable than the native Cys(6)-Cys(20) by a free-energy difference (DeltaG degrees ) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.     

Synergistic anti‐proliferative and pro‐apoptotic activity of combined therapy with bortezomib,a proteasome inhibitor,with anti‐epidermal growth factor receptor (EGFR) drugs in human cancer cells

The article to which this erratum refers was published in J Cell Physiol (2008) 216: 698–707. © 2008 Wiley‐Liss, Inc. DOI: 10.1002/jcp.21444