Richard A. Laursen

Solid-phase edman degradation: Attachment of carboxyl-terminal homoserine peptides to an insoluble resin

Attachment of peptides to insoluble supports prior to sequencing by solid-phase Edman degradation [l] has been achieved by the use of peptide amino [2] and carboxyl group [ 1,3] coupling agents. However, neither of these procedures is very useful for cyanogen bromide peptides containing C-terminal homoserine, because the former requires a side chain amino group near the C-terminus, and the latter causes side reactions of the carboxyls of aspartic and glutamic acid. Stimulated by the findings of Knobler et al. [4] and of Offord [5], we have found a simple procedure for selectively attaching cyanogen bromide peptides to resins by their C-terminal homoserine residues. The method involves lactonization of the homoserine residue with trifluoroacetic acid [6] and subsequent aminolysis of the lactone with an amino resin (fig. 1).

PROTEIN KINASE C-BETA ACTIVATES TYROSINASE BY PHOSPHORYLATING SERINE RESIDUES IN ITS CYTOPLASMIC DOMAIN

We have previously shown that protein kinase C-β (PKC-β) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993)J. Biol. Chem. 268, 11742–11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-β into nonpigmented human melanoma cells lacking PKC-β lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-β in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-β but not PKC-α was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-β with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-β. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-β and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-β. We conclude that PKC-β activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.

Solid-phase Edman degradation. The use of p-phenyl diisothiocyanate to attach lysine- and arginine-containing peptides to insoluble resins

The primary step in sequencing peptides by the solid-phase Edman degradation [l] is attachment of the peptide to an insoluble resin, generally by blocking the peptide amino groups and then activating the C-terminal carboxyl with carbonyldiimidazole [l] or a carbodiimide [2-41. A problem limiting the usefulness of the solid-phase method is that the side-chain carboxyls of aspartic and glutamic acids also become activated, with the result, at least when carbonyldiimidazole is used, that the y-carboxyl of glutamic acid becomes bound to the resin and that aspartic acid apparently forms a cyclic imide which prevents further degradation of the peptide [i] . Although procedures for selective blocking of side chain carboxyls have been devised [2], they involve a number of steps and are often unsuitable for use with nanomole amounts of peptide. A procedure we have found to be particularly useful for tryptic peptides involves attaching them by their lysine e-amino groups to aminopolystyrene usingp-phenyl diisothiocyanate. Arginine peptides are first deguanidated with hydrazine [5],

Amino acid sequence of a new type of antifreeze protein, from the longhorn sculpin Myoxocephalus octodecimspinosis

A new type of fish antifreeze protein, designated here type IV, has been isolated from the longhorn sculpin, Myoxocephalus octodecimspinosis. Sequence analysis of the protein (LS‐12) reveals that it contains 108 amino acids, is blocked at the N‐terminus by a pyroglutamyl group and has a high (17%) content of glutamine; it is thus completely unrelated to the earlier described types I, II and III fish antifreeze proteins. Circular dichroism spectra and conformational analysis based on the sequence data indicate that LS‐12 has a high helix content and probably folds as a four‐helix bundle. LS‐12 shows sequence similarity to certain plasma apolipoproteins known to have helix bundle structures, suggesting the possibility that LS‐12 may have arisen by recruitment and mutation of a plasma apolipoprotein.

Acute Experimental Allergic Encephalomyelitis in SJL/J Mice Induced by a Synthetic Peptide of Myelin Proteolipid Protein

Clinical, histologic, and ultrastructural characteristics of acute experimental allergic encephalomyelitis (EAE) induced by sensitization with a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 139-151 HCLGKWLGHPDKF were studied in SJL/J mice. Groups of mice were immunized with 20, 50, or 100 nmol of the peptide and were killed from seven to 28 days after sensitization or when they were moribund. Beginning on Day 9, the mice showed signs of EAE and the disease progressed rapidly to paralysis. Central nervous system (CNS) inflammation, edema, gliosis, and demyelination were found in all mice killed between Days 10 and 28 and white matter lesion areas correlated with clinical score at the time the mice were killed. Peripheral nerve roots and the cauda equina did not have lesions. Within the range studied, the severity of clincal or histologic disease was the same regardless of the PLP peptide dose. Two often mice immunized with 100 nmol and none of 14 mice given smaller doses of a synthetic peptide of mouse myelin basic protein (MBP) showed clinical EAE. These mice had small numbers of CNS lesions that were indistinguishable from those in PLP peptide-sensitized mice. These findings demonstrate that immunization of SJL/J mice with PLP peptide 139— 151 produces a disease with the clinical and morphologic features of CNS tissue-, whole PLP-, whole MBP-, and MBP peptide-induced acute EAE. Thus, PLP is a major encephalitogen and immune reactions to epitopes of different myelin proteins may induce identical patterns of injury in the CNS.

A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic retention of p68c-rel.

The Rel family of proteins includes a number of proteins involved in transcriptional control, such as the retroviral oncoprotein v-Rel, c-Rel, the Drosophila melanogaster developmental protein Dorsal, and subunits of the transcription factor NF-kappa B. These proteins are related through a highly conserved domain of approximately 300 amino acids, called the Rel homology domain, that contains dimerization, DNA binding, and nuclear targeting functions. Also within the Rel homology domain, there is a conserved consensus sequence (Arg-Arg-Pro-Ser) for phosphorylation by cyclic AMP-dependent protein kinase (PKA). We used linker insertion mutagenesis and site-directed mutagenesis to determine the importance of this sequence for the transformation of avian spleen cells by v-Rel and the subcellular localization of c-Rel in chicken embryo fibroblasts (CEF). The insertion of 2 amino acids (Pro-Trp) within this sequence completely abolished transformation and transcriptional repression by v-Rel and resulted in a shift in the localization of c-Rel from cytoplasmic to nuclear in CEF. When the conserved Ser within the PKA recognition sequence was replaced by Ala, there was no significant effect on transformation and transcriptional repression by v-Rel or on cytoplasmic retention of c-Rel. However, when this Ser was changed to Asp or Glu, transformation and transcriptional repression by v-Rel were significantly inhibited and c-Rel showed a diffuse nuclear and cytoplasmic localization in CEF. Although a peptide containing the recognition sequence from v-Rel can be phosphorylated by PKA in vitro, this site is not constitutively phosphorylated to a high degree in vivo in transformed spleen cells incubated with okadaic acid. Our results indicate that the transforming and transcriptional repressing activities of v-Rel and the cytoplasmic retention of c-Rel are dependent on the structure of the conserved PKA recognition motif. In addition, they suggest that phosphorylation at the conserved PKA site could have a negative effect on transformation and transcriptional repression by v-Rel and induce the nuclear localization of c-Rel.

Solid phase sequential analysis: Specific linking of acidic peptides by their carboxyl ends to insoluble resins

The success of methods such as the Edman degradation for solid-phase sequential analysis of peptides depends on having efficient methods for attaching peptides to insoluble resins before degradation. Previously described [2,3] attachment procedures using carbonyldiimidazole to couple the C-terminal carboxyl to an amino resin, suffer from the serious problem that side-chain carboxyls also become activated, leading to side reactions. In order to make the solid-phase Edman degradation generally applicable, it is essential to have a procedure for selective activation of the C-terminal carboxyl groups. We wish to report here that a.iV,N’-disubstituted carbodiimide can effect both protection of side-chain carboxyl groups and activation of the C-terminal under suitable experimental conditions (scheme A).

Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice ☆

Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in SWR/J (H-2q) and SJL/J (H-2s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In SWR/J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in SWR/J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.

Amino acid sequence of a 50 S ribosomal protein involved in both EFG and EFT dependent GTP‐hydrolysis

with a defined function are the two acidic proteins A, and A, (L, and L,,). Recently, much evidence has been accumulated that these two proteins of low molecular weight play an important role in the GTP-hydrolysis, associated with the elongation factors EFG and EFT [l-6]. Determination of the stoichiometry of ribosomal proteins indicates that 50 S ribosomes from logarith- mic growing cells contain on the average at least one copy each of A,- and AZ-protein per particle [7, 81. However, in conditions of restricted bacterial growth, under which ribosomes show an intrinsically reduced activity in protein biosynthesis [9, lo], the number of copies of A, -relative to A, -protein per ribosome decreases drastically [7, 111. In addition, particles reconstructed from “50 S cores”, supplemented with an excess of A, -protein, are significantly more active in promoting EFG- and EFT-factor mediated GTP- hydrolysis than similar particles constructed with the aid of A, -protein [ 1,6]. Since the only structural *

Isolation and characterization of an antifreeze protein from the longhorn sculpin, Myoxocephalus octodecimspinosis

A new type of antifreeze protein was isolated from the serum of the longhorn sculpin, Myoxocephalus octodecimspinosis, by gel filtration and high-performance liquid chromatography. This protein (LS-12) exhibits freezing point depression activity (thermal hysteresis) and ice crystal modification properties similar to those seen for other types of fish antifreeze polypeptide, except that ice crystals grow as hexagonal trapezohedra in the presence of LS-12, rather than hexagonal bipyramids usually seen. Ice crystal etching studies demonstrate that LS-12 does not bind to the hexagonal bipyramidal or secondary prism surfaces reported for the antifreeze polypeptides from winter flounder and shorthorn sculpin, respectively. Circular dichroism studies indicate that LS-12 has an alpha-helix content of about 60% at 1 degreesC, which is in good agreement with a value of about 70% predicted from the amino acid sequence. Limited proteolysis studies and further analysis of the amino acid sequence suggest that LS-12 consists of four amphipathic alpha-helices of similar length which are folded into a four-helix bundle. Based on its size (Mr=12299) and predicted tertiary structure, LS-12 can be regarded as the first example of a new class (type IV) of fish antifreeze protein.