The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat49–57 (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49–57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49–57 play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat49–57 in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat49–57 at cellular uptake as determined by Michaelis–Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat49–57 play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat49–57 and even to r9. Overall, a transporter has been developed that is superior to Tat49–57, protease resistent, and more readily and economically prepared.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s−1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈104 M−1 and a dissociation rate of ≥6 s−1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and Synthesis of Structurally Defined Fructan Molecules from Sucrose1

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.

Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice.

Structural features of the invariant chain fragment CLIP controlling rapid release from HLA-DR molecules and inhibition of peptide binding.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

Functional identification and reconstitution of an odorant receptor in single olfactory neurons

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Ephrin-dependent growth and pruning of hippocampal axons

Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. We report here that the ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons.

Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations

Divalent cations are thought essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the effects of 10 mM EDTA on locomotion of human blood polymorphonuclear leukocytes (PMN). In very thin slide preparations, EDTA did not impair either random locomotion or chemotaxis; motile behavior appeared to benefit from the close approximation of slide and coverslip (“chimneying”). In preparations twice as thick, PMN in EDTA first exhibited active deformability with little or no displacement, then rounded up and became motionless. However, on creation of a chemotactic gradient, the same cells were able to orient and make their way to the target, often, however, losing momentarily their purchase on the substrate. In either of these preparations without EDTA, specific antibodies to β2 integrins did not prevent random locomotion or chemotaxis, even when we added antibodies to β1 and αvβ3 integrins and to integrin-associated protein, and none of these antibodies added anything to the effects of EDTA. In the more turbulent environment of even more media, effects of anti-β2 integrins became evident: PMN still could locomote but adhered to substrate largely by their uropods and by uropod-associated filaments. We relate these findings to the reported independence from integrins of PMN in certain experimental and disease states. Moreover, we suggest that PMN locomotion in close quarters is not only integrin-independent, but independent of external divalent cations as well.

Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind α-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, d-Phe7–α-MSH4-13 analog. Structural analysis of the Re–peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate–metal–thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re–peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re–α-MSH analog. Characterization of the second-generation Re–peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of α-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties.

Synthesis and screening of small molecule libraries active in binding to DNA

Five synthetic combinatorial libraries of 2,080 components each were screened as mixtures for inhibition of DNA binding to two transcription factors. Rapid, solution-phase synthesis coupled to a gel-shift assay led to the identification of two compounds active at a 5- to 10-μM concentration level. The likely mode of inhibition is intercalation between DNA base pairs. The efficient deconvolution through sublibrary synthesis augurs well for the use of large mixtures of small, nonpeptide molecules in biological screens.

The phosphorylation state of the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cell segregation

Cell–cell recognition and patterning of cell contacts have a critical role in mediating reversible assembly of a variety of transcellular complexes in the nervous system. This study provides evidence for regulation of cell interactions through modulation of ankyrin binding to neurofascin, a member of the L1CAM family of nervous system cell adhesion molecules. The phosphorylation state of the conserved FIGQY tyrosine in the cytoplasmic domain of neurofascin regulates ankyrin binding and governs neurofascin-dependent cell aggregation as well as cell sorting when neurofascin is expressed in neuroblastoma cells. These findings suggest a general mechanism for the patterning of cell contact based on external signals that regulate tyrosine phosphorylation of L1CAM members and modulate their binding to ankyrin.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.