Biomedical subjects

A E Sneed

Publications and source records attributed to A E Sneed.

5 recordsLinked to original sources

PubMed1995

Crry and CD59 regulate complement in rat glomerular epithelial cells and are inhibited by the nephritogenic antibody of passive Heymann nephritis.

Human glomerular epithelial cells (GEC) contain CD59, decay-accelerating factor, and membrane cofactor protein. Crry is the rodent analogue to the latter two proteins. We have previously shown that the nephritogenic Ab of passive Heymann nephritis, anti-Fx1A, impairs C regulation in rat GEC. Here we examined rat GEC C regulation. 125I-labeled GEC membrane proteins were immunoprecipitated with anti-Crry, anti-CD59, or anti-Fx1A. Crry and CD59 were present in GEC. Anti-Fx1A reacted with both Crry and CD59 from GEC, as well as with purified rCrry and CD59. The alternative C pathway was studied by incubating GEC in rat serum in Mg(++)-EGTA buffer. To inhibit the function of the C regulators, anti-Crry or anti-CD59 Ab were added to GEC. Inhibition of CD59 function alone had no effect on C regulation, whereas inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. When the classical pathway of C was activated by GEC-bound IgG Ab, inhibition of either Crry or CD59 enhanced cytotoxicity, whereas inhibition of both Crry and CD59 together was additive. Therefore, Crry and CD59 are present and functionally active in GEC. Crry restricts C activation via both alternative and classical pathways. When the classical pathway of C is activated, or when Crry function is inhibited, CD59 limits C5b-9-mediated cytotoxicity. Anti-Fx1A binds to both Crry and CD59, which may account for its ability to activate the alternative pathway in vitro, and for its superior nephritogenicity in vivo.

Animals↗

PubMed1995

Molecular characterization of rat Crry: widespread distribution of two alternative forms of Crry mRNA.

The rat complement regulator 5I2 antigen/Crry is present in a widespread distribution as at least two discrete proteins of Mr 65000-70000 and 75000-85000. The molecular basis for the different proteins has not been determined. We screened a cDNA library derived from cultured rat glomerular epithelial cells with a polymerase chain reaction (PCR)-generated nucleotide probe for CR1 and Crry. Two identical clones with 1.8 kilobase inserts were obtained. Clone 6.1 consisted of 1811 nucleotides. The sequence was identical to nucleotides 42-1666 of the cDNA for rat 5I2 antigen/Crry except for 11 nucleotides at the extreme 5' and 3' ends and an exact duplication of 186 bases that would encode an additional complete short consensus repeat (SCR). By reverse transcription PCR, we show that the two forms of Crry mRNA exist in all rat cells/tissues examined. It is likely that these two Crry mRNA species differ by the absence or presence of a 186 base repeat that encodes a complete SCR. These are translated into Crry proteins, containing six and seven SCRs, respectively, which explains the different sizes of Crry proteins. The role of the SCR duplication remains to be defined.

Alternative Splicing↗

PubMed1995

Complement regulation in the rat glomerulus: Crry and CD59 regulate complement in glomerular mesangial and endothelial cells.

The complement regulators, decay accelerating factor, membrane cofactor protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined complement regulation in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES). Immunoprecipitation of 125I-labeled membrane proteins and Western blotting studies were performed with anti-Crry and anti-CD59. In both GEnC and MES, Crry was present as 53, 65, and 78 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was also present in MES, but in relatively smaller quantities. By Northern analyses, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolated rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluated next. Anti-Thy 1.1 IgG was used to activate the complement classical pathway in MES. To inhibit the function of the complement regulators, anti-CD59 and/or anti-Crry F(ab')2 antibodies were added with anti-Thy 1.1. Inhibition of Crry function led to enhanced cytotoxicity, while there was no effect when CD59 function was inhibited. The complement alternative pathway was studied by adding complement in Mg-EGTA buffer. Inhibition of Crry led to productive alternative pathway activation, which was accentuated by anti-CD59 when Crry was incompletely inhibited. Alternative pathway regulation was also evaluated in GEnC. Inhibition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEnC and MES and restricts the complement alternative pathway in both cell types. Crry also regulates the classical pathway in MES. CD59 is present and functionally active in GEnC, while it appears to have a minor role in MES.

Animals↗

PubMed1994

Molecular characterization of rat glomerular epithelial cell complement receptors.

Complement receptor type 1 (CR1) has previously been isolated from cultured rat glomerular epithelial cells (GEC) by C3b affinity chromatography. In addition, the presence of Crry in GEC and in rat glomeruli has been demonstrated. Crry appears to be the rodent analogue of human decay accelerating factor, which was previously described in human GEC and in human glomeruli. In this study, the molecular biology of these rat complement receptors is examined. A specific cDNA probe for rat CR1 was generated by reverse transcription of GEC mRNA, followed by polymerase chain reaction (PCR). The oligonucleotide primers were chosen from conserved regions spanning 271 bases in human and mouse CR1. A 271-base-pair PCR product was generated from rat GEC cDNA, the nucleotide sequence of which was 70.1% and 77.2% identical to those of the respective mouse and human sequences. This PCR product, designated rCR1-p, was then used to probe for CR1 mRNA. By northern blot analysis, rCR1-p hybridized to 4.5-kilobase (kb) mRNA from both cultured GEC and rat glomeruli and also weakly hybridized to 4.5-kb CR1 mRNA from mouse spleen. In additional northern blots, a nucleotide probe for mouse Crry hybridized to mRNA of 2.1 to 2.4 kb from rat GEC, slightly larger than the 1.9- to 2.1-kb mouse Crry mRNA. Therefore, mRNA for CR1 and Crry are present in cultured rat GEC and in rat glomeruli in vivo. To further investigate the composition of rat CR1 mRNA, northern hybridizations were performed with nucleotide probes for mouse and human CR1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of complement receptor type 1 from rat glomerular epithelial cells.

Complement receptor type 1 (C3b/C4b receptor, CR1) is known to be present in human glomerular epithelial cells (GEC) in vivo. The presence of CR1 has not been documented in rat glomeruli, although cultured rat GEC appear to express CR1 based upon their ability to rosette with complement-coated erythrocytes. In this study, we establish that CR1 is present in cultured rat GEC: (1) by isolating a 200 kDa protein from detergent-solubilized cultured rat GEC through the use of C3b affinity chromatography; (2) by Western blotting studies demonstrating reactivity of anti-human CR1 antibodies with this protein from cultured GEC; and (3) by demonstrating that C3b binding to GEC monolayers exhibits low affinity and that an estimate of the number of binding sites is 6700 per cell, both of which are comparable to that seen for CR1 in human blood cells. Furthermore, we show that CR1 is also present in rat glomeruli by Western blotting studies with anti-human CR1. Anti-human CR1 also identifies a 70 kDa protein from cultured GEC and isolated glomeruli. This 70 kDa protein is likely to be the CR1-like protein, designated Crry, which was initially identified in the mouse and has significant homology to human CR1. Crry may be present in rat GEC instead of decay accelerating factor, which is present in human GEC.

Animals↗