Biomedical subjects
Publications and source records attributed to R J Folz.
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In order to characterize the function of the COOH-terminal (c) region of eukaryotic signal peptides, a 14-amino acid long segment was deleted from a secreted rat liver and intestinal protein, preapolipoprotein-A-IV. This deletion spanned the c-region plus all potential signal peptidase cleavage sites. The functional consequences of this mutation were assessed using an in vitro transcription/translation/microsomal membrane processing system. Removal of these residues had no effect on interaction of the nascent preprotein with signal recognition particle as measured by a translational arrest assay. Although no signal peptidase cleavage of the mutant was detected, the efficiency of its co-translational translocation was similar to the wild type protein. A postinitiation translocation assay was utilized to compare the translocation capabilities of nascent wild type and mutant proteins as their chain lengths were progressively increased. No difference was detected between the two species suggesting that their initial conformations are functionally equivalent as measured by the translocation machinery. We conclude from these studies of preapolipoprotein-A-IV that (i) the efficiency of translocation is not dependent on signal peptidase cleavage and (ii) structural features present in the c-region of the signal peptide are not necessary for interaction with signal recognition particle or for subsequent targeting to, and translocation across, microsomal membranes.
We have previously described a novel mutant of human preproapolipoprotein A-II (pre(delta pro)apoA-II) in which the wild-type 18-amino acid-long signal sequence (Gly18 decreases) was functionally redefined to 20 amino acids in length (Ala 20 decreases). We have used this mutant as a model preprotein to probe the substrate specificity of eukaryotic signal peptidase. Site-saturation mutagenesis was performed resulting in the substitution of 13 different amino acids (acidic, basic, aromatic, hydrophobic, and small-neutral) for Ala20 (or position -1). The effects of these substitutions were assessed using an in vitro transcription/translation/microsomal membrane processing system. NH2-terminal sequence analysis of the 13 mutant proteins demonstrated that amino acids which occupy position -1 in a signal peptide are critical in establishing a good context for signal peptidase cleavage and that two or more potential sites of cleavage may compete for recognition by signal peptidase.
A new algorithm is presented which can be used to examine the physical-chemical properties of amino acids at sites of co- or post-translational processing. This algorithm has been incorporated into a computer program known as PARA-SITE. Thirty different parameters can be studied for amino acids which occupy comparable positions in naturally occurring proteins. PARA-SITE should aid in the design and interpretation of protein engineering experiments which seek to dissect structure/activity relationships.
The two principal protein components of human high density lipoprotein particles, apoA-I and apoA-II, are initially synthesized as prepropolypeptides. The function of their NH2-terminal prosegments is not known. We have previously shown that deletion of the pentapeptide prosegment (Ala-Leu-Val-Arg-Arg) from human preproapoA-II redirects signal peptidase cleavage to a site located between the second and third residues of the mature protein (Folz, R. J., and Gordon, J. I. (1986) J. Biol. Chem. 261, 14752-14759). The hexapeptide prosegment of human preproapoA-I differs from other NH2-terminal propeptides in that it terminates with paired glutamine residues (Arg-His-Phe-Trp-Gln-Gln). To examine its role in the early events of protein export, we isolated a full-length human preproapoA-I cDNA and deleted its propeptide coding region by oligonucleotide-directed mutagenesis. The effects of this deletion on co-translational translocation and signal peptidase processing were assessed using an in vitro transcription/translation/microsomal membrane processing system. Propeptide deletion reduces the efficiency of co-translational translocation/processing in both reticulocyte and wheat germ lysates but does not affect the fidelity of signal peptidase cleavage. This reduced efficiency does not appear to reflect differences in signal recognition particle-nascent protein interaction as measured by a translational arrest assay. However, differences in translocation/processing rates were noted in a postinitiation translocation/cleavage assay. This assay employed the initiation inhibitor edeine to generate nascent wild type and mutant proteins of increasing chain length which were subsequently presented to canine pancreatic membranes. Unlike preproapoA-I, pre(delta pro)apoA-I retained its ability to undergo translocation and proteolytic processing even after the entire protein was synthesized. These data suggest that the propeptide in human preproapoA-I may play a role in promoting an optimal structure for co-translational translocation and processing.
The human insulin-like growth factor I (IGF-I) gene can specify two primary translation products, IGF-IA and IGF-IB, which differ at their COOH termini and which must undergo extensive proteolytic processing to produce the mature 70-residue circulating peptide. We have used an in vitro transcription/translation/microsomal membrane processing system to define the early events in IGF-I biosynthesis. Our results demonstrate that protein synthesis is initiated at the first in-frame methionine codon for both human IGF-IA and IGF-IB mRNAs, yielding primary translation products of 153 and 195 residues, respectively. Both are cotranslationally translocated into the lumen of canine pancreatic microsomes. Cotranslational proteolytic processing results in the removal of an unusually large 48-amino acid signal peptide. Computer-assisted sequence analysis revealed that this prepeptide contains two domains. Its COOH-terminal 19 residues possess features which are typically encountered in eukaryotic signal peptides. The rest of the IGF-I signal sequence is represented by an unusual "extended" NH2-terminal domain containing an overall net charge of +5 and 3 clustered cysteines. The size and charge of this NH2-terminal domain distinguish it from comparable regions of other eukaryotic signal peptides including the presegment of human IGF-II. Although no specialized biological role can currently be ascribed to this unique NH2-terminal domain, its remarkable sequence conservation between human and rat suggests an as yet unknown functional significance.
Computer programs are presented which incorporate 2 different algorithms for predicting the site of signal peptide cleavage for eukaryotic preproteins. These programs can be used to identify sites of signal peptidase cleavage of putative preproteins to facilitate the design and interpretation of signal peptide mutagenesis experiments, and to engineer artificial prepolypeptides.
The functions of NH2-terminal propeptides are not known. We have used apoA-II as a model to study prosegment structure/function relationships. The primary translation product of human apolipoprotein A-II mRNA contains an 18-amino acid signal peptide, a 5-amino acid propeptide, and the mature 77-amino acid plasma protein sequence. Its propeptide was deleted by site-directed mutagenesis of a cloned cDNA. The effects of this mutation on cotranslational translocation and proteolytic processing were assessed using an in vitro transcription/translation/microsomal membrane processing system. Deletion of the propeptide did not affect cotranslational translocation. However, without its propeptide, signal peptidase cleavage was redirected to a different site located between the 2nd and 3rd residues of the mature protein. Since the primary structure of the signal peptide was not altered in the mutant, these results suggest that sequences located downstream from the signal peptidase cleavage site (e.g. in propeptides) may modulate, or participate in defining, the correct site of cotranslational proteolytic processing.
N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2) and exhibited 1.2% of the binding affinity, 2.4% of the biological potency, and 30% of the maximum activity of the native hormone. Preliminary fluorescence microscopy demonstrated binding of N alpha-biotinyl-N epsilon-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein-labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)N epsilon-acetimidoglucagon, prepared via a manual Edman degradation of N epsilon-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2). The second residue, serine, has also been removed. Semisynthetic coupling of alternative residues to such derivatives will provide insight into the role of the amino-terminal residues in mediating the biological actions of the hormone.