Cyclic modular beta-sheets.

The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (

Increase of expression and activation of chemokine CCL15 in chronic renal failure.

Chemokines are believed to be involved in the pathogenesis of chronic renal failure (CRF). In CRF, significantly increased CCL15-IR plasma concentrations were detected. Whereas in plasma of healthy individuals one predominant CCL15-IR molecule with a M(w) of 15kDa [high molecular weight (HMW-CCL15-IR)] was identified, CRF plasma contains increased concentrations of truncated CCL15-IR molecules [intermediate molecular weight (IMW-CCL15-IR)]. HMW-CCL15-IR isolated from hemofiltrate revealed an M(w) of 10141.3, corresponding to deglycosylated CCL15(1-92) carrying a N-terminal pyrrolidone carboxylic acid. CCL15(12-92) was identified as a major component of IMW-CCL15-IR in CRF plasma. Compared to CCL15(1-92), in monocytes CCL15(12-92) causes stronger induction of intracellular calcium flux, chemotactic activity, and adhesion to fibronectin. Intracellular calcium flux assays revealed that, in comparison to peripheral blood mononuclear cells (PBMC) of healthy donors, PBMCs of CRF patients demonstrated an increased sensitivity to CCL15. Our results point to an involvement of the CCL15-CCR1 axis in the pathophysiology of CRF.

Identification of NH(2)-terminal amino acid residues essential for the biological activity of leukotactin-1.

Leukotactin-1 (Lkn-1), a human CC chemokine that binds to both CC chemokine receptor (CCR)1 and CCR3, is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2)-terminus. Serial deletion studies showed that at least three amino acid residues, alanine-alanine-aspartic acid (A-A-D), preceding the first cysteine at the NH(2)-terminus are essential for the biological activity of Lkn-1. Point mutation and deletion studies for the three amino acids were performed in the present study. Substitutions of the first alanine residue with other amino acids did not cause significant loss of biological activities. Deletion of the third amino acid, aspartic acid, resulted in more than 100-fold loss of the activity. Deletion of two amino acids, alanine-alanine (A-A) or alanine-aspartic acid (A-D), resulted in almost complete loss of the activity. Loss of agonistic activity by deletion of two amino acids was due to impaired binding to CCR1. These results identify that alanine-aspartic acid residues preceding the first cysteine at the NH(2)-terminus are essential for the binding and biological activity of Lkn-1.

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC-2, a high affinity CCR1 ligand.

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.

Unmasking ligand binding motifs: identification of a chemokine receptor motif by NMR studies of antagonist peptides.

Determining the critical structural features a ligand must possess in order to bind to its receptor is of key importance to the understanding of vital biological processes and to the rational design of small molecule therapeutics to modulate receptor function. We have developed a general strategy for determining such ligand binding motifs using low temperature NMR structures of peptides with the desired receptor binding properties. This approach has been successfully applied to determine a binding motif for the chemokine receptor CXCR4. The motif identified provides a detailed guide for the design of small molecule antagonists against CXCR4, which are much sought after to aid in the treatment of a number of conditions including human immunodeficiency virus type 1 infection and a variety of cancers.

Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha.

The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.

Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells.

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2alphabeta, hence is designated as MIP-2gamma. Mouse MIP-2gamma was identified by electrocloning and is highly homologous to human MIP-2gamma. Northern blotting revealed that MIP-2gamma was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2gamma was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2gamma expression by monocyte-derived DC. Recombinant MIP-2gamma from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2gamma is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2gamma does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.

Solution structure of murine macrophage inflammatory protein-2.

The solution structure of murine macrophage inflammatory protein-2 (MIP-2), a heparin-binding chemokine that is secreted in response to inflammatory stimuli, has been determined using two-dimensional homonuclear and heteronuclear NMR spectroscopy. Structure calculations were carried out by means of torsion-angle molecular dynamics using the program X-PLOR. The structure is based on a total of 2390 experimental restraints, comprising 2246 NOE-derived distance restraints, 44 distance restraints for 22 hydrogen bonds, and 100 torsion angle restraints. The structure is well-defined, with the backbone (N, Calpha, C) and heavy atom atomic rms distribution about the mean coordinates for residues 9-69 of the dimer being 0.57 +/- 0.16 A and 0.96 +/- 0.12 A, respectively. The N- and C-terminal residues (1-8 and 70-73, respectively) are disordered. The overall structure of the MIP-2 dimer is similar to that reported previously for the NMR structures of MGSA and IL-8 and consists of a six-stranded antiparallel beta-sheet (residue 25-29, 39-44, and 48-52) packed against two C-terminal antiparallel alpha-helices. A best fit superposition of the NMR structure of MIP-2 on the structures of MGSA, NAP-2, and the NMR and X-ray structures of IL-8 are 1.11, 1.02, 1.27, and 1.19 A, respectively, for the monomers, and 1.28, 1.10, 1.55, and 1.36 A, respectively, for the dimers (IL-8 residues 7-14 and 16-67, NAP-2 residues 25-84). At the tertiary level, the main differences between the MIP-2 solution structure and the IL-8, MGSA, and NAP-2 structures involve the N-terminal loop between residues 9-23 and the loops formed by residues 30-38 and residues 53-58. At the quaternary level, the difference between MIP-2 and IL-8, MGSA, or NAP-2 results from differing interhelical angles and separations.

Accessibility of selenomethionine proteins by total chemical synthesis: structural studies of human herpesvirus-8 MIP-II.

The determination of high resolution three-dimensional structures by X-ray crystallography or nuclear magnetic resonance (NMR) is a time-consuming process. Here we describe an approach to circumvent the cloning and expression of a recombinant protein as well as screening for heavy atom derivatives. The selenomethionine-modified chemokine macrophage inflammatory protein-II (MIP-II) from human herpesvirus-8 has been produced by total chemical synthesis, crystallized, and characterized by NMR. The protein has a secondary structure typical of other chemokines and forms a monomer in solution. These results indicate that total chemical synthesis can be used to accelerate the determination of three-dimensional structures of new proteins identified in genome programs.

Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines.

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to the macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7%) and MIP-1alpha (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes, T-lymphocytes and, to a lesser degree, eosinophils at nanomolar concentrations; it has no effect on neutrophil migration. In receptor-binding assays, MIP-5 shows IC50 values of 12 nM for competition with 125I-MIP-1alpha for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for binding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 was only able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3.

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.

Cloning and characterization of a novel murine macrophage inflammatory protein-1 alpha receptor.

We have cloned a novel CC chemokine receptor cDNA from mouse thymus. The deduced amino acid sequence shows 74% identity to the human monocyte chemotactic protein (MCP)-1 receptor (CC CKR-2b) and 54% to a recently cloned murine macrophage inflammatory protein (MIP)-1alpha receptor (Gao, J. L., and Murphy, P. M.(1995) J. Biol. Chem. 270, 17494-17501). Northern blot analysis of mouse tissues showed that the mRNA was also expressed in heart, spleen and liver, and to a lesser extent in lung and brain. The rank order of CC chemokine competition for 125I-labeled human RANTES (regulated on activation, normal T-cell expressed and secreted) binding to human embryonic kidney (HEK) 293 cells stably transfected with the receptor cDNA was murine MIP-1alpha >> human MIP-1beta > human RANTES > murine RANTES > murine MIP-1beta > human MCP-2 > murine MCP-1 (JE) > human MIP-1alpha > human MCP-3 > human MCP-1. Of the chemokines tested, only murine MIP-1alpha, human and murine MIP-1beta and RANTES, human MCP-2, and JE were able to induce mobilization of intracellular Ca2+ from fura-2-loaded HEK 293 cells expressing the receptor. These results suggest that this receptor functions as a high affinity murine MIP-1alpha receptor; however, it is likely to be an important target for the biological activities of several CC chemokines in mouse.

Heteronuclear (1H, 13C, 15N) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer.

A full high-resolution three-dimensional solution structure of the monocyte chemoattractant protein-1 (MCP-1 or MCAF) homodimer has been determined by heteronuclear multidimensional NMR. MCP-1 is a member of a family of small proteins which play a crucial role in immune surveillance by orchestrating the recruitment of specific leukocytes to areas of immune challenge. The protein was uniformly isotopically enriched with 13C and 15N by expression in Escherichia coli, and complete sequence-specific resonance assignments were obtained by a combination of heteronuclear double- and triple-resonance experiments. The secondary structure was deduced from characteristic patterns of NOEs, 13 C alpha/beta chemical shifts, measurements of 3JHNH alpha scalar couplings, and patterns of slowly exchanging amide protons. Because MCP-1 forms symmetrical homodimers, additional experiments were carried out to unambiguously establish the quaternary contacts. NOEs from these novel experiments were merged with more traditional heteronuclear separated NOE measurements in an iterative strategy to partition the restraints between explicit inter/intrasubunit contacts and a class wherein both were retained as ambiguous. With more than 30 restraints per residue, the three-dimensional structure is well-defined with a backbone rmsd of 0.37 A to the mean over residues 5-69 of the dimer. We compare the structure with those recently reported for the related chemokines MIP-1 beta and RANTES and highlight the differences in terms of receptor specificity and function as well as interpret the known biological activity data of MCP-1 mutants.

BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties.

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.

Proton NMR assignments and solution conformation of RANTES, a chemokine of the C-C type.

1H NMR has been used to investigate the structural properties of RANTES, a protein from the C-C branch of the chemotactic cytokine family that has a strong chemoattractive effect on monocytes, lymphocytes, and eosinophils. Titration of pH from 5.0 to 2.5 indicates that RANTES is extensively aggregated in solution above pH 4.0. At pH 3.7 the protein is mostly dimeric, although this species does dissociate to the monomer with a Kd of 35 microM. NMR data have been acquired and resonance assignments made for the dimeric species. Structures of the dimer have been generated by distance geometry and simulated annealing calculations that utilized 1956 intramolecular distance restraints, 120 intermolecular distance restraints, 164 dihedral angle restraints, and 68 restraints enforcing 34 hydrogen bonds (17.0 restraints per residue). The structure is well-defined (average root mean square deviation from the average structure of 0.38 +/- 0.06 and 0.53 +/- 0.12 A for backbone heavy atoms of residues 4-66 of the monomer and dimer, respectively). Each monomer consists of a C-terminal alpha-helix packing against a three-stranded antiparallel beta-sheet and two short N-terminal beta-strands; dimerization occurs between the N-terminal regions of each monomer. This quaternary structure is very different from that of the C-X-C chemokines such as interleukin-8 and melanoma growth stimulatory activity but similar to that found for the C-C chemokine macrophage inflammatory factor 1 beta. Distinct structural differences between RANTES and other chemokines at both the tertiary and quaternary level are discussed with regard to the distinct biological functions of the C-C and C-X-C members of this protein family.

Three-dimensional structures of alpha and beta chemokines.

Members of the chemokine family of proteins play a key role in the orchestration of the immune response. This family has been further divided into two subfamilies, alpha and beta, based on sequence, function, and chromosomal location. To date, the three-dimensional structures of two members of the alpha subfamily, interleukin-8 (IL-8) and platelet factor 4, and one member of the beta subfamily, human macrophage inflammatory protein-1 beta (hMIP-1 beta), have been solved by either NMR or X-ray crystallography. In this review, we discuss their three-dimensional structures and their possible relationship to function. The structures of the monomers are very similar, as expected from the significant degree of sequence identity between these proteins. The quaternary structures of the alpha and beta chemokines, however, are entirely distinct and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1 beta dimer is elongated and cylindrical. Platelet factor 4 is a tetramer comprising a dimer of dimers of the IL-8 type. The IL-8 dimer comprises a six stranded anti-parallel beta-sheet, three strands contributed by each subunit, on top of which lie two antiparallel helices separated by approximately 14 A, and the symmetry axis is located between residues 26 and 26' (equivalent to residue 29 of hMIP-1 beta) at the center of strands beta 1 and beta 1'.(ABSTRACT TRUNCATED AT 250 WORDS)

MIP-1 gamma: molecular cloning, expression, and biological activities of a novel CC chemokine that is constitutively secreted in vivo.

We have identified a novel CC chemokine family member, herein termed MIP-1 gamma in view of its similarity to existing members of the MIP-1 group. The murine protein has a predicted length of 100 amino acids. Like MIP-1 alpha, recombinant MIP-1 gamma acts as a pyrogen when administered intracerebroventricularly. MIP-1 gamma and MIP-1 alpha engage the same high-affinity receptor on neutrophils, activating calcium release within seconds following cell contact. Pretreatment with either chemokine abolishes responses to the other, and to itself, suggesting utilization of a common signaling pathway. However, unlike MIP-1 alpha or any of the other CC chemokines, MIP-1 gamma is expressed constitutively by a wide variety of tissues, and circulates in the blood of healthy mice at concentrations of approximately 1 microgram/ml (90 nM). It would therefore be predicted that MIP-1 gamma occupies most of the CC chemokine receptors that exist in the intravascular compartment. As such it might, under normal circumstances, markedly influence responses to the inducible CC chemokines.

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.

Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry.

During the analysis of an Asp-N digest of a recombinant hematopoietic growth factor by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we observed pseudomolecular ions corresponding to reduced forms of peptides known to be present only in single disulfide linkages. Chromatographic fractionation of the peptide digest, followed by MALDI-MS and electrospray ionization (ESI) MS, confirmed that the reduced peptides were not present in the map. Fragmentation of the disulfide-linked peptides into their reduced forms occurred upon ionization from different matrices (alpha-cyano-4-hydroxycinnamic acid,2,5-dihydroxybenzoic acid, and in some instances sinapinic acid) but only after increasing the laser fluence to above threshold. Analysis of the disulfide-linked peptide fractions by ESI-MS, before and after mixing and drying with matrix, indicated that the matrix did not cause reduction. In a low-energy tandem mass spectrometric experiment with one of the cystinyl peptides, fragmentation did not occur preferentially at the disulfide bond. The pseudomolecular ions exhibited the same m/z values by MALDI-MS as their chemically reduced counterparts, indicating that they arose due to prompt fragmentation or "in-source decay" rather than "post-source decay". This finding is important for MALDI-MS analysis of peptide maps of proteins and peptide fractions with intact disulfides.

Hepatocyte growth factor and macrophage inflammatory protein 1 beta: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.

T-cell migration into tissue depends on a cascade of rapid and selective adhesive interactions with endothelium. "Triggering" is a step in that cascade required to activate T-cell integrins. Hepatocyte growth factor (HGF) may be a physiologically relevant trigger, since we demonstrate that HGF can induce both adhesion and migration of human T-cell subsets and can be detected immunohistochemically on inflamed endothelium. HGF preferentially induces responses from T cells of memory phenotype, in contrast to macrophage inflammatory protein 1 beta (MIP-1 beta), a chemokine which acts preferentially on naive cells. HGF, like the chemokines, binds to heparin, and HGF retained in extracellular matrix is efficient in promoting migration. Further, both MIP-1 beta and HGF induce actin polymerization within seconds, kinetics that approach those required to contribute to physiologic triggering. HGF is a member of a structural family distinct from the chemokines, whose only known receptor is the tyrosine kinase c-Met. HGF induces tyrosine phosphorylation on T cells apparently via a distinct receptor, since no c-Met is detectable by surface staining, PCR, or anti-phosphotyrosine immunoprecipitation. Thus, promotion of T-cell adhesion and migration are previously undescribed functions of HGF that we propose are relevant to selective T-cell recruitment.