Molecular cloning and characterization of a cDNA encoding the human leucocyte vacuolar protein sorting (h1Vps45).

We have isolated a novel cDNA clone from human leucocyte cDNA library, encoding a Sec1p-like vacuolar protein sorting (h1Vps45) which is believed to be implicated in vesicular transportation. Although the deduced amino acid (AA) sequence of this cDNA has revealed 97% identity to other known mammalian vacuolar protein sorting, there is an extensive variation in nucleotide sequence in comparison to that of three previously reported human (hVps45), rat (rVps45) and mouse (mVps45) vacuolar protein sorting (Vps45) cDNAs [1-3]. At the nucleotide sequence level h1Vps45 demonstrated 90% homology to the hVps45 and rVps45 and 89% identity to mVps45 with no significant homology in their noncoding regions. The 2.4 Kb mRNA corresponding to the h1Vps45 clone is widely distributed in a variety of human tissues expressing highest levels in peripheral blood mononuclear cells (PBMC), neutrophils, heart, spleen, and testis. The chromosomal mapping studies have demonstrated that the h1Vps45 is localized to long arm of human chromosome 1 at q21-q22. Our data indicates that we have isolated, characterized and mapped a novel cDNA encoding h1Vps45, which may play an important role in protein trafficking as well as have clinical significance in the release of inflammatory mediators e.g. histamine, bradykinin and cytokine release.

Purification and characterization of a human bradykinin binding protein from inflammatory cells.

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

High affinity bradykinin binding to human inflammatory cells.

Specific direct bradykinin (BK) binding and competitive inhibition was detected in human neutrophil and peripheral blood mononuclear cell (PBMC) detergent solubilized extracts and purified plasma membranes using in vitro radioreceptor ligand binding. Scatchard analyses of [125I]-BK binding revealed an equilibrium dissociation constant (Kd) of 2.9 x 10(-11) M for neutrophils and 5.6 x 10(-11) M for PBMC using [des-arg9]-BK a B1 agonist; 2.6 x 10(-11) M for neutrophils, 6.2 x 10(-11) M for PBMC with BK a B2 agonist; 5.4 x 10(-11) M for PBMC using Lys-BK a B2 agonist. The number of binding sites (Bmax) was calculated to be 0.113 fM/microgram protein (720 receptors per cell) for neutrophils and 0.200 fM/microgram protein (1289 receptors per cell) for PBMC with the B1 agonist while with the B2 agonists the values were 0.128 fM/microgram protein (818 receptors per cell) for neutrophils and 0.157 fM/microgram protein (1005 receptors per cell) for PBMC with BK, and 0.293 fM/microgram protein (1870 receptors per cell) with Lys-BK for PBMC. In a competitive binding inhibition assay using neutrophil and PBMC glycerol purified plasma membranes, high affinity binding in the nanomolar range was detected to Lys-BK and BK but with [des-arg9]-BK a 10-100 fold lower order affinity was observed this being indicative of pharmacologically defined B2 characteristics.

Polymorphism of IgE gene in chronic urticaria.

Our aim was to determine whether heterogeneity of the IgE (C epsilon) gene could be demonstrated in patients with chronic urticaria (CU). We performed Southern blots on DNA extracted from peripheral blood leucocytes of 20 patients with CU and 20 normal controls. Using a human C epsilon gene probe containing four exons of the constant segment of the IgE heavy chain, we showed the presence of a restriction fragment length polymorphism of the C epsilon gene segment in four of 20 patients with CU, but in none of 20 normal subjects. Family studies of two propositi revealed the presence of this C epsilon gene polymorphism in other family members. Our data show that a proportion of patients with CU have a polymorphism of the constant segment of the C epsilon gene. Further studies of this polymorphic gene fragment indicated that it was derived from duplication of the 3rd and 4th exons of functional C epsilon gene and was very likely to be located close to this gene. It raises the possibility that polymorphism of the functional C epsilon gene may affect expression of this gene. This could possibly lead to dysfunctional IgE-receptor interaction with consequent alteration in mediator release.

A preparative method for sequencing proteins and peptides: in situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes.

A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.

Purification of histamine receptor proteins from detergent-solubilized human peripheral blood mononuclear cells.

Histamine is released from mast cells and basophils by either immunological or nonimmunological mechanisms. Histamine, which is the most potent short acting mediator released from these cells, exerts its diverse biological actions by binding to cell surface histamine receptors. We report the affinity purification of histamine receptor proteins from Triton X-100 solubilized peripheral human blood mononuclear cells which include lymphocytes and monocytes. Three different designs of histamine affinity columns were constructed; all three resulted in the same material being eluted. This consisted of bands which on SDS-PAGE after boiling and reduction had the following molecular weights: 193K, 84K, 58K, 48K, 37K, and 16K. The most abundant bands were of molecular weights 193K, 48K, and 16K, and these were disulfide bonded together to form a high molecular weight complex. (The 58K band was present in lower amounts than the others, and in only a few fractions. It had the same molecular weight as the dimeric form of histamine methyltransferase which is present in small amounts in mononuclear cells and may therefore have copurified.) The histamine binding proteins described in this report were purified by conventional affinity chromatography, rather than by an expression cloning approach which obviates the use of any protein chemistry. Consequently, we had the advantage of being able to verify the histamine binding specificity of our purified proteins directly and with several independent assays as follows. The histamine binding specificity of all three columns was established by specific elution with histamine, by preabsorption of crude cell extract with excess free histamine prior to column application, and by comparison with control columns. Independent determination of the binding specificity, using a radioreceptor dot blot assay, of the eluate containing only the 193K, 48K, and 16K disulfide-linked subunits confirmed that the purified material bound specifically to [3H]histamine and that a 300-500-fold degree of purification from tissue extract had been obtained. Following cell surface radioreceptor cross-linking of radiolabeled histamine to intact mononuclear cells, the 16K band was detected, indicating it to be the ligand-binding subunit for histamine. These same three proteins were purified from T lymphocyte and monocytoid cell lines, indicating that both lymphocyte and monocyte subsets of mononuclear cells express these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10.

We previously reported the purification and partial amino acid sequence of a novel murine cytokine designated CP-10, which has chemotactic activity for murine polymorphonuclear cells (PMN) and macrophages. The complete cDNA encoding an 88-amino acid polypeptide has been isolated and the sequence is presented here. Transient transfection of CP-10 cDNA into CV-1 cells confirmed the chemotactic activity of rCP-10 for murine PMN. CP-10 has sequence homology with members of the S100 family of Ca(2+)-binding proteins with pronounced amino acid sequence similarities within the putative N- and C-terminal Ca(2+)-binding sites, but differences within their connecting hinge and C-terminal regions. We have confirmed the hypothesis of Kligman and Hilt that functional specificity of individual members of the S100 protein family may reside in the hinge region. A synthetic peptide corresponding to the hinge region of CP-10 (CP-10(42-55) was compared with native CP-10 in chemotaxis and skin test assays. Native CP-10 had potent activity for phagocytic cells, but not lymphocytes, in vitro (optimal activity, 10(-11) to 10(-13) M) and elicited a sustained recruitment of neutrophils and mononuclear cells over 24 h in vivo. The hinge-region peptide had strong chemotactic activity for murine phagocytic cells (optimal activity, 10(-10) - 10(-11) M) but elicited only a transient infiltration of neutrophils over 4 to 8 h after intradermal injection. Results indicate that although the hinge region contributes significantly to the functional specificity of the S100 protein CP-10, sustained cellular recruitment typical of a delayed type hypersensitivity response is apparently dependent on the structural integrity of the protein.

A genomic clone encoding the alpha chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules.

LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa beta subunit but are distinguished by their alpha chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. We report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in lambda phage Charon 4A and subsequent "rescue" of the lambda phage. Transfection with the purified recombinant lambda DNA yielded a transfectant that expressed the three human alpha chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine beta chain.

Double 9;22 translocation with hyperdiploidy appearing in blastic transformation of chronic granulocytic leukemia.

Chromosome studies in a 26-year-old female with Ph1-positive chronic granulocytic leukemia showed the development of both hyperdiploid and tetraploid cell lines in the blastic transformation phase. An additional 9;22 translocation and additional chromosomes No. 8, 11, 13 and 22 were found in the hyperdiploid cell line. This unusual finding suggests that the hyperdiploidy may have developed from misdivisions in the tetraploid cell line rather than by the more accepted mechanism of nondisjunction or selective endoreduplication from a diploid cell.

C-banding studies in patients with Ph1+ chronic granulocytic leukaemia.

The bone marrow chromosomes of 15 patients with Ph1+ chronic granulocytic leukaemia were studied, using both G- and C-banding. In all cases the Ph1 chromosome was formed by the translocation between chromosomes 9 and 22 and the material from chromosome 22 was found to be translocated randomly onto one or other of the pair of 9 chromosomes. Preliminary results suggest that when the translocation was on the 9 chromosome having a smaller C-band, additional abnormalities occurred in blastic transformation, whereas when the 9 chromosome with the larger C-band was involved in the translocation, additional abnormalities were not found in blastic transformation. These observations require confirmation from a larger series. C-banding also showed that there was a greatly increased heteromorphism of the C-band areas of the chromosome pair 9 in this disease, and an increased heteromorphism in the C-bands of chromosome pair 1, when compared with a control group.