Differential processing of human and rat E1 α precursors of the branched-chain α-keto acid dehydrogenase complex caused by an N-terminal proline in the rat sequence

The N-terminal sequences of the E1 α, E1β and E2 subunits of the human branched-chain α-keto acid dehydrogenase complex have been determined by microsequencing. The N-termini of human E1β and E2 subunits (Val and Gly, respectively) are indentical to those of the corresponding rat and bovine subunits. However, the N-terminus of the human E1 α subunit (Ser) is identical to bovine, but differs from the rat E1 α (Phe0 subunit. Comparison of the N-terminal sequences of human and rat E1 α subunits shows that the serine residue at the + 1 position in the human sequence is replaced by a proline residue in the rat sequence. The presence of the proline residue apparently causes a 5′-shift by one residue in the cleavage site by the mitochondrial processing peptidase in the rat sequence, when compared to the human sequence. The results provide evidence that the mitochondrial processing peptidase cannot cleave an X-pro bond, similar to trypsin, chymotrypsinand microsomal signal peptidases.     

Molecular cloning and functional expression of the human sodium channel beta1B subunit, a novel splicing variant of the beta1 subunit.

The voltage gated sodium channel comprises a pore-forming alpha subunit and regulatory beta subunits. We report here the identification and characterization of a novel splicing variant of the human beta1 subunit, termed beta1B. The 807 bp open reading frame of the human beta1Beta subunit encodes a 268 residue protein with a calculated molecular mass of 30.4 kDa. The novel human beta1B subunit shares an identical N-terminal half (residues 1-149) with the human beta1 subunit, but contains a novel C-terminal half (residues 150-268) of less than 17% sequence identity with the human beta1 subunit. The C-terminal region of the human beta1B is also significantly different from that of the rat beta1A subunit, sharing less than 33% sequence identity. Tissue distribution studies reveal that the human beta1Beta subunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle. Functional studies in oocytes demonstrate that the human beta1B subunit increases the ionic current when coexpressed with the tetrodotoxin sensitive channel, NaV1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human beta1 and rat beta1A subunits.     

Evidence for the spontaneous formation of interspecies hybrid molecules of human, rat and bovine serum albumins

Utilizing electrophoretic and gel filtration techniques it was shown that a bovine C-terminal peptic fragment [residues 307-582] spontaneously forms interspecies hybrid molecules with three complementary N-terminal fragments derived from human [residues 1-308; 49-308] and rat [residues 1-308] albumins. The fragments associate with 1:1 stoichiometry to produce an albumin-like complex which has a molecular weight and electrophoretic mobility similar to intact albumin. These data demonstrate, for the first time, that albumin fragments derived from different species associate in a complementary fashion and provide direct evidence that the tertiary structure may be highly conserved.     

The amino acid sequence of a type I copper protein with an unusual serine- and hydroxyproline-rich C-terminal domain isolated from cucumber peelings.

We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N-terminal 110 amino acid-long domain shows 30-37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid-long C-terminal domain rich in 4-hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence.     

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.     

Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage.

Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C-terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ.     

Processing of proapolipoprotein AI requires specific conformation

Apolipoprotein AI of human high-density lipoproteins is secreted by hepatocytes as a proapolipoprotein with a N-terminal hexapeptide sequence (Arg-His-Phe-Trp-Gln-Gln-) which differs from the prosequence of rat apolipoprotein AI (Trp-Asp-Phe-Trp-Gln-Gln). The two proteins have in common the unusual cleavage site -Gln-Gln-Asp-Glu-. It is hydrolysed by a specific serum proteinase with the release of mature apo AI. We synthesized a model substrate for the study of the final processing of pro-apo AI by the serum proteinase. It is an undecapeptide embracing the human pro-hexapeptide sequence and the first five N-terminal residues of apo AI, covalently linked to a hydrophilic resin. The N-terminal arginine residue was 3H-labelled. [formula; see text] This sequence was not cleaved by human serum under the conditions under which rat serum processes the pro-form of apo AI secreted by rat hepatocytes. Pepsin and chymotrypsin fragmented the undecapeptide at sites characteristic for these proteinases. We conclude that the proteolytic cleavage at the specific site (-Gln-Gln-Asp-Glu-) requires the correct conformation in addition to the specific amino-acid sequence.     

Differences between ranamargarin and other tachykinins in the stimulation of ion transport in frog skin

In frog skin, tachykinins stimulate ion transport by interaction with NK1-like receptors. The structural requirements of the peptide are the presence of the C-terminal sequence Phe-X-Gly-Leu-Met-NH₂ and at least one Pro residue in the N-terminal sequence. In this paper, we demonstrate that the C-terminal amino acid must be amidated but it can be different from Met, and that the sequence cannot be longer or shorter than 11–12 amino acids. Unexpectedly, Ranamargarin (14 amino acids, no Pro residue) increased the short circuit current value by 48 ± 0.3%. On the basis of considerable experimental evidence, we suggest that Ranamargarin interacts with a receptor different from those of other tachykinins.     

Biochemical Characterization of Recombinant Human Nerve Growth Factor

Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.     

Alternative processing pathways for preprovasoactive intestinal peptide in the enteric nervous system of the rat

In order to study biosynthetic processing of preprovasoactive intestinal peptide (prepro VIP) we have raised antisera to sequences that flank the biologically active peptides VIP and PHI (peptide with N-terminal His and C-terminal Ile). We have used these antisera in radioimmunoassays to identify the N-terminal flanking peptide (NFP) and C-terminal flanking peptide (CFP)-like immunoreactivities in rat brain and gastrointestinal tract. Concentrations of NFP-LI were similar to those of VIP in brain and throughout the gut. Concentrations of CFP-LI were 10-20% those of VIP-LI but could be increased 5-fold by digestion with carboxypeptidase B, suggesting that the C-terminal lysine residue of prepro VIP is not normally removed during processing. In rat stomach the NFP-LI was of higher molecular weight and greater hydrophobicity than the intestinal component. The data are consistent with alternative processing pathways for prepro VIP in enteric nerves of rat stomach and intestine.     

Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.     

Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.     

Human spleen histone H1. Isolation and amino acid sequences of three minor variants, H1a, H1c, and H1d

Following the previous determination of the main variant H1b of human spleen histone H1, we have determined the complete amino acid sequence of another variant, H1d. Limited chymotryptic digestion of H1d produced four fragments, I to IV, and one partial fragment I-II, as in the case of H1b. These fragments were aligned with two overlapping peptides, produced by another enzyme from the intact H1d. We also confirmed the C-terminal sequence of H1d by carboxypeptidase digestion. This H1d has an acetylated N-terminal serine, equimolar alanine or valine residue at 17, and is composed of 212 residues. The molecular weight was 21,233 for the alanine variant and 21,261 for the valine variant in the unmodified form. We also deduced the total sequences of H1a and H1c in a similar way, considering the maximum homology with H1b and H1d. Each N-terminal serine residue is acetylated, too. H1a consists of 222 amino acid residues and has a molecular weight of 22,178 in its unmodified form; the H1c consists of 220 residues and has a molecular weight of 22,218 in that form. The human spleen H1 sequences varied to about the same extent in the N-terminal 40 and C-terminal 110 residues. However, the sequences of the about 70 internal residues are well conserved between the variants. The extent of differences among the human H1 variants is similar to, or rather smaller than, those among the mammalian somatic H1 species. The implications of these differences in the sequence for H1 function are discussed from the evolutionary viewpoint.     

The unique N-terminal sequence of testis angiotensin-converting enzyme is heavily O-glycosylated and unessential for activity or stability.

The testis-specific isozyme of angiotensin-converting enzyme (ACE) is identical, from residue 68 to the C terminus, to the second half or C-terminal domain of somatic ACE. However, the first 67 residues, comprising the signal peptide and a Ser-/Thr-rich 36-residue sequence that constitutes the N terminus of mature testis ACE, are unique. We have expressed a mutant human testis ACE lacking this 36-residue N-terminal sequence and find that compared to the wild-type protein the mutant is 15 kDa smaller due to the loss of greater than 90% of all O-linked sugars, but that it retains full enzymatic activity and is stable in culture. Heavy O-glycosylation is a property of testis ACE that is not shared by the somatic enzyme and is attributable to this unique sequence.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

A novel form of the polypeptide PHI isolated in high yield from bovine upper intestine. Relationships to other peptides of the glucagon-secretin family

A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser- Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF).     

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1-14 and Δ1-24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association.     

Differences in Action of PACAP-27 and PACAP-38 on Guinea Pig Gallbladder Smooth Muscle Using Synthetic C-terminally Modified PACAP Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na⁺ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.     

In vitro T-cell immunogenicity of oligopeptides derived from the region 92-110 of the 16-kDa protein of Mycobacterium tuberculosis

The 16-kDa protein of Mycobacterium tuberculosis provokes specific immune responses; it is thus a target for the development of peptide-based diagnostic reagents and subunit vaccines. Previous studies have demonstrated the presence of several regions containing murine and human T-cell epitopes. Within the 91-110 immunodominant domain, we found that peptides comprising the sequence of 91SEFAYGSFVRTVSL104 elicit specific T-cell responses in both human T-cell clones and human peripheral blood mononuclear cells (PBMC) from PPD+ (purified protein derivative) individuals. Elongation of this peptide towards the C-terminal end did not provide more effective peptides, but the removal of residue 91Ser resulted in an almost complete loss of functionality. However, the introduction of an acetyl group at the N-terminal of residue 92Glu produced a shorter peptide (Ac-92EFAYGSFVRTVSL104) exhibiting properties required for efficient T-cell responses. CD measurements indicated that peptide 91SEFAYGSFVRTVSLPVGADE110 adopts a helical conformation in trifluoroethanol. We found that the N-terminal part of this sequence plays a major role in the induction of proliferative T-cell responses and is responsible for the highly ordered, helical secondary structure. The "lead" structure described here could also be considered in the development of synthetic peptides or multicomponent peptide mixtures for the early detection, monitoring, or preventing Mycobacterium tuberculosis infection with optimized T-cell response-provoking capacity.