Identification of a C-reactive protein binding site in two hepatic carboxylesterases capable of retaining C-reactive protein within the endoplasmic reticulum.

C-reactive protein (CRP) is normally synthesized by hepatocytes at relatively low rates and is retained within the endoplasmic reticulum (ER) via interaction with two carboxylesterases (termed gp60a and gp60b), which themselves are restricted to the ER by their COOH-terminal retention signals (HIEL and HTEL). During the acute phase response, an increase in CRP synthesis is accompanied by a decrease in its ER retention as a result of a decrease in the CRP binding affinity of gp60b. Our previous data indicated that the esterase active site, the CRP binding site, and the ER retention signal are functionally distinct. In the present studies, we have identified CRP-binding peptides produced by proteolytic cleavage of gp60a. The sequence shared by two CRP-binding peptides indicated that the CRP binding site of gp60a is contained within residues 477-499. These results were confirmed by expression of cDNAs coding for gp60a and b as bacterial fusion proteins. Fusion proteins containing the complete esterase COOH terminus bound CRP, whereas those truncated at residue 477 (or the homologous site in gp60b) did not. Based on the known crystal structures of three homologous hydrolases, the putative CRP-binding site of the gp60s is located on the surface and is physically distant from the esterase active site and the COOH-terminal ER retention signal.

Novel putative protein kinase clones from a rat large granular lymphocyte tumor cell line.

Protein kinases have been implicated in a number of regulatory mechanisms including signal transduction in many cells. To address the possibility that the large granular lymphocyte (LGL) also uses one or more unique protein kinases for LGL functions, an efficient method was developed to obtain partial cDNA clones for putative protein kinases from a rat LGL tumor cell line, RNK. Using the polymerase chain reaction (PCR), one hundred and nine amplified DNA segments were cloned and sequenced from a RNK cDNA library. One hundred and eight of these segments were putative protein kinases based on their deduced sequences. Among these were nine putative protein kinases, seven of which were putative tyrosine kinases and two were putative serine kinases. Only four of the putative kinases identified in this study were identical, or nearly identical, to previously published protein kinases. The deduced amino acid sequences of the remaining clones differed by seven or more amino acid residues in the amplified segments from all known protein kinase, and were considered to be novel putative protein kinases. Two of the five novel putative kinases showed lymphoid-restricted patterns of expression on Northern analyses. The method described in this paper provides an efficient cloning strategy for novel protein kinases, and is similar to that published in a previous report. Although the PCR primers used in this report differ only slightly from those in the previous report, we used a much higher annealing temp (50 degrees C) than in the previous report (37 degrees C), which may in part account for our higher yield of putative protein kinase sequences.

Molecular cloning of rat cytolysin.

Rat cytolysin is one of the cytolytic factors present in the cytoplasmic granules of rat NK-like cytolytic cells and purified cytolysin exhibits an apparent Mr or 70 kDa. Cytolysis produced by cytolysin occurs in the presence of Ca2+ and is accompanied by the formation of membrane lesions of 160 A diameter. We have isolated a cDNA encoding rat cytolysin from the cDNA library of a rat large granular lymphocyte (LGL) cell line, by hybridization of the rat library with a cDNA probe for mouse perforin. The amino acid sequence deduced from the nucleotide sequence of the isolated cDNA insert indicates that the mature cytolysin protein consist of 534 amino acids with a leader peptide of 20 amino acids. The protein contains two functionally important domains: the first domain is believed to contain the transmembrane channel and the second domain consists of an epidermal growth factor-type "class B" cysteine-rich region. A comparison with mouse perforin indicates that the two genes are very similar (89.9% nucleotide and 84.9% amino acid identity). Northern blot hybridization analysis indicates that cytolysin mRNA is expressed in rat lymphocytes (lymphokine-activated killer cells and LGL cells) and LGL cell lines.

Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines.

The protein portion of the immunosuppressive glycoprotein uromodulin is identical to the Tamm-Horsfall urinary glycoprotein and is synthesized in the kidney. Evidence that the glycoproteins are the same is based on amino acid sequence identity, immunologic cross-reactivity, and tissue localization to the thick ascending limb of Henle's loop. Nucleic acid sequencing of clones for uromodulin isolated from a complementary DNA bank from human kidney predicts a protein 639 amino acids in length, including a 24--amino acid leader sequence and a cysteine-rich mature protein with eight potential glycosylation sites. Uromodulin and preparations of Tamm-Horsfall glycoprotein bind to recombinant murine interleukin-1 (rIL-1) and human rIL-1 alpha, rIL-1 beta, and recombinant tumor necrosis factor (rTNF). Uromodulin isolated from urine of pregnant women by lectin adherence is more immunosuppressive than material isolated by the original salt-precipitation protocol of Tamm and Horsfall. Immunohistologic studies demonstrate that rIL-1 and rTNF bind to the same area of the human kidney that binds to antiserum specific for uromodulin. Thus, uromodulin (Tamm-Horsfall glycoprotein) may function as a unique renal regulatory glycoprotein that specifically binds to and regulates the circulating activity of a number of potent cytokines, including IL-1 and TNF.

Inhibition of cytolysin activity in large granular lymphocyte granules by lipids: evidence for a membrane insertion mechanism of lysis.

The ability of various compounds to inhibit the lytic activity of purified cytoplasmic granules from LGL tumors was tested. The lytic activity of granule cytolysin was modestly inhibited by inorganic phosphate, and various monophosphoesters, with I50 values in the range of 8-20 mM. Among monophosphoesters, choline phosphate was exceptionally potent, with an I50 of 1.4 mM. In contrast to the inhibition by phosphate esters, the parent compounds such as neutral sugars, glycerol, and choline, showed no detectable inhibition at 50 mM. A lysolipid bearing a short aliphatic chain and some detergents were inhibitory with I50 values in the range of 1 mM. Lysolipids with longer aliphatic chains, phospholipids as liposomes, and related lipid compounds were found to display potent inhibition of the hemolytic activity of LGL granule cytolysin, with I50 values in the range of 0.2-30 microM. Soluble globular proteins inhibited LGL granule cytolysin hemolytic activity with I50 of 0.07-0.4 mg/ml. However, lipoproteins were 2-3 orders of magnitude more potent inhibitors, with L50 values of less than 1 microgram/ml. The observed potent inhibition by lipid compounds lends support to a model of the lytic action of granule cytolysin in which soluble cytolysin molecules insert into the membrane lipid bilayer during the course of the lytic event.

Cytolytic and biochemical properties of cytoplasmic granules of murine lymphokine-activated killer cells.

The incubation of murine spleen cells in the lymphokine interleukin 2 (IL 2) gives rise to lymphokine-activated killer (LAK) cells capable of lysing fresh tumor cells in short-term lytic assays. During the course of cultures used to generate LAK cells, cytoplasmic granules were prepared and were analyzed for the presence of the cytolysin previously described in large granular lymphocytes (LGL) and cytotoxic T lymphocytes (CTL). Such cytolysin activity is initially undetectable, appears after 2 days of culture, and continues to increase until day 7. The LAK cytolysin has properties similar to those of previously described cytolysins with respect to nonspecific killing of various target cells, rapid kinetics, and absolute dependence on calcium. Antibodies raised against purified LGL tumor granules neutralized the activity of the LAK cytolysin. The precursors of both the LAK cells and the cells bearing the cytolysin are eliminated by treatment with anti-asialo-GM1 and complement, strongly suggesting that the actual LAK effector cells and the cytolysin-bearing cells are identical. Biochemical analysis of the LAK granules indicate that they contain the lysosomal enzyme arylsulfatase. The protein content of granules isolated from various days of culture with r-IL 2 undergoes a dramatic change, with major protein bands around 30,000 daltons becoming prominent, as well as the cytolysin protein band at 70,000 daltons. These data suggest that the mechanism of cell lysis by LAK cells is similar to that of CTL and natural killer-mediated lysis, and each of these forms of lymphocyte-mediated cytolysis is based on a granule exocytosis mechanism.

Apical fibrocavitary lesions of the lung in rheumatoid arthritis. Report of two cases and review of the literature.

Two patients with rheumatoid arthritis and fibrocavitary lesions in the upper lobes of the lungs are described. Postmortem pathologic studies of the lungs revealed the presence of clinically unsuspected necrobiotic nodules with cavitation and excluded other possible causes such as infections and vasculitis. These findings support the view that apical fibrocavitary disease is a clinically distinct pattern of lung involvement in rheumatoid arthritis.

Heterotopic ossification presenting as arthritis.

We report 4 patients with arthritis as a presenting manifestation of heterotopic ossification. This cause of arthritis has only recently been noted. The synovial fluids from all 4 patients showed low leukocyte counts (90, 670, 167 and 500/mm3) while the protein concentrations were discordantly high in 3 cases (3.1, 5.3, 4.3 and 2.6 g/dl, respectively). The mechanism for the relatively elevated synovial protein concentration is unknown. These findings may be useful for the early diagnosis of heterotopic ossification.