Identification of haptoglobin as an alternative ligand for CD11b/CD18.
ABSTRACT Haptoglobin is an acute phase protein with presumed anti-inflammatory activities. We report that purified fluorescein-labeled haptoglobin 1-1 binds to THP1 and U937 promonocytic cell lines, to monocytes, to granulocytes, and to a subset of CD8+ T cells and to NK cells. Studies with radioiodinated haptoglobin on THP1 cells were consistent with specific binding to one class of receptors with a density of 1.7 x 10(5) binding sites per cell and a low affinity of 6.5 x 10(-6) Kd. Binding was increased by Ca2+ and by Ca2+ and ADP. Binding to THP1 and U937 cells could be inhibited by preincubation with nonfluoresceinated haptoglobin and by fibrinogen, but not by albumin, transferrin, or alpha1-acid glycoprotein. Fibrinogen binds to the CD11b/CD18 integrin. We therefore examined whether haptoglobin has the same receptor. The anti-CD11b mAb44 indeed inhibited the binding of fluoresceinated haptoglobin to THP1 and U937 cell lines, and haptoglobin inhibited the binding of the anti-CD11b mAb anti-Leu15 and mAb44 to both cell lines. An anti-CD18 mAb partly inhibited the binding of fluoresceinated haptoglobin to THP1 and U937, indicating that the beta-chain of MAC-1 is also involved in haptoglobin binding. There was no interference between the binding of anti-CD4, anti-CD11a, or anti-CD11c mAb and haptoglobin binding to THP1 cells. Binding of haptoglobin to purified CD11b/CD18 indicates that it binds directly to the receptor. Haptoglobin is an alternative low affinity ligand for the CD11b/CD18 integrin, suggesting that this acute phase protein might regulate MAC-1-dependent cell function in vivo.
- SourceAvailable from: Pingping Jiang[Show abstract] [Hide abstract]
ABSTRACT: Membranous nephropathy (MN), a common cause of idiopathic nephrotic syndrome in adults, remains a potentially devastating problem worldwide. At present, there is no reliable noninvasive method for predicting and/or monitoring this glomerular disease, and its pathophysiology remains poorly understood. In the present study, the urinary proteome profile of rats after 10 days of an induction of passive Heymann nephritis (PHN), which resembles human MN, was compared to that of the baseline (control) urine prior to the induction of PHN by anti-Fx1A injection. Each pool of PHN and control urine samples (n = 10 each) was labeled with different fluorescent dyes (Cy3 or Cy5), and equal amounts of the labeled proteins of both pools were resolved in the same 2D gel, together with an internal standard labeled with Cy2. Two-dimensional difference gel electrophoresis revealed a number of protein spots whose expression levels were altered during PHN. Eighteen protein spots with >1.5-fold changes and p < 0.05 were selected for subsequent identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. They were successfully identified as serum albumin precursor, alpha-1-antitrypsin, preprohaptoglobin, liver-regeneration-related protein, and transthyretin (which increased during PHN) and E-cadherin, MPP7, tropomyosin beta, kallikrein, and alpha-2u globulin (which decreased in the PHN urine). Among these proteins, the increase in urinary preprohaptoglobin has particularly drawn our attention because of its byproduct, haptoglobin (Hp), which is involved in the protection of tissue damage from hemoglobin-induced oxidative stress. Western blotting and enzyme-linked immunosorbent assay clearly showed a markedly increased level of Hp in the urine, but not in the serum, of the PHN animals. Our findings may lead to a significant advance in the attempt to define a new therapeutic target and/or novel biomarker for human MN.Journal of Proteome Research 09/2007; 6(8):3313-20. · 5.06 Impact Factor
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ABSTRACT: Peritoneal endometriotic tissues synthesize and secrete haptoglobin (pHp), which has an analogous nucleotide sequence to hepatic haptoglobin found in serum (sHp). This study performed enzymatic digestions and lectin binding assays to determine if differences in protein glycosylation exist between sHp and pHp, which may provide insight into pHp function and/or identify epitopes for development of novel methods of medical management of endometriosis. To reduce the dependence on surgical collection of peritoneal tissues from women, recombinant peritoneal Hp (rpHp) was produced and its glycosylation analyzed for future functional studies. These results showed the apparent molecular weight of pHp was 3 kDa smaller than sHp. Desialylation and complete N-deglycosylation elicited similar shifts in sHp and pHp electrophoretic migration, suggesting similar sialic acid content and indicating the 3 kDa variance was due to carbohydrate content, not protein degradation, respectively. Sequential deglycosylation of the four sHp N-glycan chains caused a 3 kDa shift per N-glycan removed suggesting the 3 kDa difference between sHp and pHp may be one N-glycan chain. Lectin ELISA and lectin-blotting analyses demonstrated increased pHp and rpHp interactions with MAL and LTL but no difference in binding to SNL compared to sHp from healthy individuals, identifying variations in the ratios of alpha(2-3) to alpha(2-6) sialic acid and fucose residues. Recombinant pHp was 100-fold over-expressed with a similar glycosylation pattern to pHp, albeit in an unprocessed alpha-beta Hp polypeptide form. These results are the first to identify differences between pHp and sHp glycosylation and lay groundwork further studies to characterize anomalies in glycan composition and structure, which likely impart pHp with known immunomodulatory functions and may be used as epitopes for development of immune based therapeutics for novel, non-surgical management of endometriosis.Glycoconjugate Journal 02/2002; 19(1):33-41. · 1.88 Impact Factor
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ABSTRACT: The present study was undertaken to investigate correlation between some hematological parameters, acute phase proteins and immunoglobulins in the experimentally infected goats with Besnoitia caprae from the time of infection till 360 days post infection (DPI). Six male goats, approximately 12–16 months old, were inoculated subcutaneously with approximately 1.3 × 108 bradyzoites of B. caprae and blood samples were collected at weekly intervals from the jugular vein of the goats. Total leukocyte count and differential leukocyte counts were determined. Acute phase proteins (APPs) including serum amyloid A (SAA), haptoglobin (Hp), fibrinogen and ceruloplasmin were undertaken at weekly intervals. We evaluated an enzyme-linked immunosorbent assay (ELISA) (using a somatic antigen of bradyzoite) to detect anti-B. caprae antibodies in caprine sera. Cysts were present in the skin biopsies of the distal parts of the leg of the infected goats from 28 DPI. From 30 to 360 DPI, results showed that the APPs concentrations including SAA, Hp, fibrinogen and ceruloplasmin were enhanced in the serum of infected goats. However, there were some variation in hematological parameters; the differences were not significant with those of the normal values. Some variations were seen in the levels of specific antibodies against this parasite and they had correlation with some hematological parameters and acute-phase proteins.Journal of parasitic diseases