Microtubules-associated intracellular localization of the NH2-terminal cellular prion protein fragment

By utilizing double-labeled fluorescent cellular prion protein (PrPC), we revealed that the NH2-terminal and COOH-terminal PrPC fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells and HpL3-4, a hippocampal cell line established from prnp gene-ablated mice [Nature 400 (1999) 225]. Of note, the NH2-terminal PrPC fragment, which predominantly localized in the intracellular compartments, congregated in the cytosol after the treatment with a microtubule depolymerizer (nocodazole). Truncated PrPC with the amino acid residues 1-121, 1-111, and 1-91 in mouse (Mo) PrP exhibited a proper distribution profile, whereas those with amino acid residues 1-52 and 1-33 did not. These data indicate the microtubules-associated intracellular localization of the NH2-terminal PrPC fragment containing at least the 1-91 amino acid residues.     

Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor

The goal of this study was to investigate the molecular interaction between superantigens and the T-cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T-cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing Vβ3, Vβ12, Vβ13.2, Vβ14, Vβ15, Vβ17 and Vβ20. SEC2 and SEC3 have the additional ability to expand T cells bearing Vβ13.1, and their expansion of Vβ3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate Vβ3 and Vβ13.1 was determined by the residue in position 26, located in the base of the SEC α3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a Vβ expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co-ordinates of a recently reported murine TCR β-chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T-cell receptor. In this model complex, the complementarity-determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the Vβ element contact SEC3 predominantly through residues in the α3 cavity of the toxin. CDR3 of the β chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of Vβs 3 and 13.1 by the SEC superantigens.     

Effect of peptides--structural analogs of NH2-terminal sites of fibrin alpha- and beta-chains--on specific binding of the NH2-terminal disulfide bond of fibrin with fibrinogen

Peptides Gly-Pro-Arg-Pro and Gly-His-Arg-Pro (fibrin alpha- and beta-chain NH2-terminal analogs, respectively) are studied for their effect on fibrinogen (F) and fibrin NH2-terminal disulphide knot (N-DSK) specific binding. Both peptides are found to inhibit the formation of soluble and insoluble F-N-DSK-complexes through inhibition of the interdomain D-E-binding. Gly-Pro-Arg-Pro is much more potent inhibitor than Gly-His-Arg-Pro. Lowering the insoluble F-N-DSK-copolymer quantity by concentration-dependent way these peptides do not change its composition described by the formula [F(N-DSK)2]n. Invariability of fibrinogen and N-DSK copolymer structure is asserted. In this structure neighbouring fibrinogen molecules are bound by two N-DSK molecules via the D1-E1 and D2-E2 binding sites.     

Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.     

Immunochemical evidence for the binding of caldesmon to the NH2-terminal segment of actin

The binding of caldesmon and its actin-binding fragments to actin was studied by using peptide antibodies directed against two actin sites implicated in actomyosin interactions. Antibodies against residues 1-7 on skeletal alpha-actin strongly inhibited the binding of caldesmon to actin and perturbed to a smaller extent the interaction between actin and the actin binding fragments. Carbodiimide coupling of ethylenediamine to the NH2-terminal acidic residues on actin inhibited the binding of caldesmon and its fragments to actin to a similar extent as the (residues 1-7) antibodies. Antibodies against residues 18-28 showed only limited competition with caldesmon for the binding to actin. These results lead to the following conclusions. (i) The NH2-terminal residues on actin play an important role in the binding of caldesmon to actin, (ii) residues 18-28 on actin do not form a major caldesmon interaction site, and (iii) the actin-binding fragments do not contain the full actin-binding interface. These conclusions and other literature data suggest that caldesmon regulates the actomyosin ATPase by competing with myosin.ATP for the NH2-terminal segment on actin.     

The NH2-terminal extension of rat liver arginyl-tRNA synthetase is responsible for its hydrophobic properties

Rat liver arginyl-tRNA synthetase is found in extracts either as a component (Mr = 72,000) of the multienzyme aminoacyl-tRNA synthetase complex or as a low molecular weight (Mr = 60,000) free protein. The two forms are thought to be identical except for an extra peptide extension at the NH2-terminus of the larger form which is required for its association with the complex, but is unessential for catalytic activity. It has been suggested that interactions among synthetases in the multienzyme complex are mediated by hydrophobic domains on these peptide extensions of the individual proteins. To test this model we have purified to homogeneity the larger form of arginyl-tRNA synthetase and compared its hydrophobicity to that of its low molecular weight counterpart. We show that whereas the smaller protein displays no hydrophobic character, the larger protein demonstrates a high degree of hydrophobicity. No lipid modification was found on the high molecular weight protein indicating that the amino acid sequence itself is responsible for its hydrophobic properties. These findings support the proposed model for synthetase association within the multienzyme complex.     

NH2-terminal cleavage of xenopus fibroblast growth factor 3 is necessary for optimal biological activity and receptor binding.

Fibroblast growth factor 3 (FGF3) was originally identified as the mouse proto-oncogene Int-2, which is activated by proviral insertion in tumors induced by mouse mammary tumor virus. To facilitate the biological characterization of the ligand, we have analyzed its homologue in Xenopus laevis, XFGF3. Here we confirm that the X. laevis genome contains two distinct FGF3 alleles, neither of which is capable of encoding the NH2-terminally extended forms specified by the mouse and human FGF3 genes. Unlike the mammalian proteins, XFGF3 is efficiently secreted as a Mr 31,000 glycoprotein, gp31, which undergoes proteolytic cleavage to produce an NH2-terminally truncated product, gp27. Processing removes a segment of 18 amino acids immediately distal to the signal peptide that is not present in the mammalian homologues. By inserting an epitope-tag adjacent to the cleavage site, we show that a substantial amount of the gp27 is generated intracellularly, although processing can also occur in the extracellular matrix. Two residues are also removed from the COOH terminus. To compare the biological properties of the different forms, cDNAs were constructed that selectively give rise to the larger, gp31, or smaller, gp27, forms of XFGF3. As judged by their ability to cause morphological transformation of NIH3T3 cells, their mitogenicity on specific cell types, and their affinity for the IIIb and IIIc isoforms of Xenopus FGF receptors, gp27 has a much higher biological activity than gp31. Sequence comparison revealed an intriguing similar cleavage motif immediately downstream of the signal peptide cleavage site in the NH2-terminus of mouse and human FGF3. Analysis of secreted mutant mouse FGF3 confirmed an additional NH2-terminal processing at the corresponding sequence motif. NH2-terminal trimming of Xenopus and mammalian FGF3s may therefore be a prerequisite of optimal biological activity.     

Immunohistochemical localization of the NH2-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH(2)-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH(2)-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH(2) antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH(2)-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.     

Physical and chemical properties of the NH2-terminal glutamic acid and lysine forms of human plasminogen and their derived plasmins with an NH2-terminal lysine heavy (A) chain.

Comparative physical and chemical data are described for the human NH2-terminal Glu-plasminogen and Lys-plasminogen forms in order to determine the exact relationship between these two types of the zymogen. The molecular weights of Glu-plasminogen and Lys-plasminogen were similar and were determined to be 83, 800 plus or minus 4, 500 and 82, 400 plus or minus 3, 300, respectively, by sedimentation equilibrium methods. The molecular weights were identical in dodecyl sulfate solutions, approximately 83, 000, by sedimentation equilibrium methods. The sedimentation coefficients, s-020, w of Glu-plasminogen and Lys-plasminogen were determined to be 5.0 S, and 4.4 S, respectively. These two plasminogen forms had different partial specific volumes, and calculations of the frictional coefficients from sedimentation coefficients and molecular weights indicated conformation differences. Glu-plasminogen appeared to be larger in size than Lys-plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. The amino acid compositions of Glu-plasminogen and Lys-plasminogen, and their major isolated isoelectric forms, were found to be similar, but several amino acid residues (glutamic acid, alanine, isoleucine, phenylalanine, and lysine) were found to be significantly higher in the Glu-plasminogen forms. The derived plasmins from both the Glu- and Lys-plasminogens with an nh2-terminal Lys- heavy (A) chain were found to have identical molecular weights of 76, 500 plus or minus 2, 500, and sedimentation coefficients, s-020, w of 4.3 S.     

Association of the S-100-related calpactin I light chain with the NH2-terminal tail of the 36-kDa heavy chain

Calpactin I, a Ca2+- and phospholipid-binding cytoskeletal protein, which serves as a major substrate of protein-tyrosine kinases, was isolated from bovine intestine and lung as a species containing two 36-kDa heavy chains and two 10-kDa light chains. The heavy chain is comprised of two distinct domains which can be identified by limited proteolysis: a COOH-terminal 33-kDa core, which contains the Ca2+- and phospholipid-binding sites, and an NH2-terminal tail, which contains the major site of phosphorylation by pp60v-src. To determine the site of association of the light chain on the heavy chain, we analyzed the association states of the light chain, core, and tail by sucrose gradient centrifugation after limited chymotryptic digestion. The core was not detected in higher Mr complexes with the light chain, and the tail cosedimented with a light chain dimer. The tail, isolated from chymotryptic digests and radiolabeled with 125I, was found to form a specific complex with the light chain, but not the core. The authentic tail and a synthetic peptide corresponding to residues 1-29 of the calpactin I heavy chain were both able to specifically inhibit the reassociation between heavy and light chain, whereas a synthetic peptide corresponding to residues 15-33 was inactive. These results suggest that the tail may serve as a site of regulation by light chain or phosphorylation.     

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.     

NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes

Hepatopoietin A (HPTA) is an acidic heparin-binding polypeptide growth factor for hepatocytes with properties distinct from other known heparin-binding growth factors. HPTA is a heterodimer consisting of a heavy and a light polypeptide chain with Mr of 70,000 and 35,000 respectively. HPTA is a complete mitogen for hepatocytes in that it stimulates DNA synthesis in hepatocytes maintained in serum-free medium. Its complete purification from rabbit serum or human plasma was reported by us elsewhere (R. Zarnegar and G. Michalopoulos, 1989). In the present communication we report the N-terminal amino acid sequence of the HPTA light chain up to 24 residues (VVNGKPTRTNVGRMVSLKYRNKHI) and show that this sequence is unique and not related to any other proteins or growth factors based on computer search analysis. We have also raised antiserum against a synthetic peptide corresponding to the sequence of N-terminal amino acids residues 1 to 24, which recognizes the whole HPTA molecule. This antiserum as well as oligonucleotide probes corresponding to the N-terminal amino acid sequence of HPTA can be used as probes to identify tissue(s) of origin of this growth factor and assist in molecular cloning of its gene.     

Relaxin structure. Quasi allosteric effect of the NH2-terminal A-chain helix

The NH2-terminal heptapeptide in the relaxin A-chain (Arg-Met-Thr-Leu-Ser-Glu-Lys) has been replaced by chemical means with three different helix-promoting peptides (Arg-Met-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala, and the insulin segment Gly-Ile-Val-Glu-Gln). The partially protected NH2 terminally shortened relaxin derivative (N epsilon A16,N epsilon B8-bis(methyl-sulfonylethyloxycarbonyl)des-ArgA1,MetA2 , ThrA3,LeuA4,SerA5,GluA6,LysA7-B29-relaxin) has been prepared by a combination of cyanogen bromide digestion and Edman degradation of the epsilon-amino-protected derivative followed by mixed anhydride coupling with the synthetic peptides. All three derivatives have been isolated and purified by high performance liquid chromatography. Whole relaxins shortened at the NH2 terminus of the A-chain by 4 or more amino acid residues are biologically inactive in the mouse pubic symphysis assay (Büllesbach, E. E., and Schwabe, C. (1986) Biochemistry 25, 5998-6004). The introduction of the artificial peptides causes significant biological activity to reappear (about 30%). The loss of structural integrity of relaxins shortened by 4 or more residues of the A-chain NH2 terminus as observed by circular dichroism spectroscopy is largely reversed by the addition of the synthetic peptides. Our results suggest that no single amino acid in the NH2-terminal region of the A-chain is functionally important but that the presence of a helix is required for biological activity.