CD22 ligand binding regulates normal and malignant B lymphocyte survival in vivo

The CD22 extracellular domain regulates B lymphocyte function by interacting with alpha2,6-linked sialic acid-bearing ligands. To understand how CD22 ligand interactions affect B cell function in vivo, mouse anti-mouse CD22 mAbs were generated that inhibit CD22 ligand binding to varying degrees. Remarkably, mAbs which blocked CD22 ligand binding accelerated mature B cell turnover by 2- to 4-fold in blood, spleen, and lymph nodes. CD22 ligand-blocking mAbs also inhibited the survival of adoptively transferred normal (73-88%) and malignant (90%) B cells in vivo. Moreover, mAbs that bound CD22 ligand binding domains induced significant CD22 internalization, depleted marginal zone B cells (82-99%), and reduced mature recirculating B cell numbers by 75-85%. The CD22 mAb effects were independent of complement and FcRs, and the CD22 mAbs had minimal effects in CD22AA mice that express mutated CD22 that is not capable of ligand binding. These data demonstrate that inhibition of CD22 ligand binding can disrupt normal and malignant B cell survival in vivo and suggest a novel mechanism of action for therapeutics targeting CD22 ligand binding domains.     

A structure-function study of ligand recognition by CD22beta

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.     

A snapshot of the Ixodes scapularis degradome

Parasitic encoded proteases are essential to regulating interactions between parasites and their hosts and thus they represent attractive anti-parasitic druggable and/or vaccine target. We have utilized annotations of Ixodes scapularis proteases in gene bank and version 9.3 MEROPS database to compile an index of at least 233 putatively active and 150 putatively inactive protease enzymes that are encoded by the I. scapularis genome. The 233 putatively active protease homologs hereafter referred to as the degradome (the full repertoire of proteases encoded by the I. scapularis genome) represent ~1.14% of the 20485 putative I. scapularis protein content. Consistent with observations in other animals, the content of the I. scapularis degradome is ~6.0% (14/233) aspartic, ~19% (44/233) cysteine, ~40% (93/233) metallo, ~28.3% (66/233) serine and ~6.4% (15/233) threonine proteases. When scanned against other tick sequences, ~11% (25/233) of I. scapularis putatively active proteases are conserved in other tick species with ≥ 60% amino acid identity levels. The I. scapularis genome does not apparently encode for putatively inactive aspartic proteases. Of the 150 putative inactive protease homologs none are from the aspartic protease class, ~8% (12/150) are cysteine, ~58.7% (88/150) metallo, 30% (45/150) serine and ~3.3% (5/150) are threonine proteases. The I. scapularis tick genome appears to have evolutionarily lost proteolytic activity of at least 6 protease families, C56 and C64 (cysteine), M20 and M23 (metallo), S24 and S28 (serine) as revealed by a lack of the putatively active proteases in these families. The overall protease content is comparable to other organisms. However, the paucity of the S1 chymotrypsin/trypsin-like serine protease family in the I. scapularis genome where it is ~12.7% (28/233) of the degradome as opposed to ~22-48% content in other blood feeding arthropods, Pediculus humanus humanus, Anopheles gambiae, Aedes Aegypti and Culex pipiens quinquefasciatus is notable. The data is presented as a one-stop index of proteases encoded by the I. scapularis genome.     

A snapshot of the Chinese SOL Project

In 2003, the International Solanaceae Project (SOL) was initiated by an international consortium of ten countries including Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy and the United States. The first major effort of the SOL aimed to produce a DNA sequence map for euchromatin regions of 12 chromosomes of tomato (Solanum lycopersicum) before 2010. Here we present an update on Chinese effort for sequencing the euchromatin region of chromosome 3.     

Human basophils express CD22 without expression of CD19.

BACKGROUND:Even modern automatic cell counters cannot count basophils precisely. Therefore, we need a rapid, accurate, precise, and easy method for counting basophils. METHODS:Using flow cytometry, basophils (CD22+/CD19-) and B cells (CD22+/CD19+) were counted. Within a large lymphocyte light scatter gate, % basophils (G%baso) and % B cells (G%B) were determined from the total count. Another method of analysis was to make two regions (R1 for basophils and R2 for B cells) and to determine in those the % basophils (R1%baso) and % B cells (R2%B) without gating. The flow cytometric basophil counts of the blood of 21 normal controls and 43 chronic myelogenous leukemia (CML) patients were compared with manual basophil count (Ma%baso) and basophil count by Coulter electronic cell counter (Hialeah, FL) (Auto%baso). CD22+/CD19- cells were sorted by a FACSCalibur (Becton Dickinson, San Jose, CA). RESULTS:The G%baso of all samples was 4.66 +/- 5.35%, and R1%baso was 4.23 +/- 4.88%, and they were well-correlated (r = 0.996, P < 0.001). The G%B of all samples was 1.55 +/- 1.68%, and R2%B was 1.59 +/- 1.67%, and they were also well-correlated (r = 0.993, P < 0.001). Their correlation was better in normal controls than in CML. G%baso was well-correlated to Ma%baso (r = 0.827) and Auto%baso (r = 0.806), and R1%baso was well-correlated to Ma%baso (r = 0.831) but showed poor correlation to Auto%baso (r = 0.734). Auto%baso revealed the poorest correlation to Ma%baso (r = 0.692). The sorted CD22+/CD19- cells were all basophils (99.48 +/- 0.30%), and they revealed CD13, CD33, and dim CD45 expression, whereas CD3, CD14, CD16, and HLA-DR were not detected on them. CONCLUSIONS: We discovered a specific marker combination to identify basophils (CD22+/CD19-), and we suggest that flow cytometric analysis using these markers is an easy, reliable, and accurate method of basophil counting.     

Molecular mechanism of a novel CD59‐binding peptide sp22 induced tumor cells apoptosis

Some short peptides discovered by phage display are found to be able to inhibit cancer growth and induce cancer cell apoptosis. In this study, a novel cancer‐targeting short peptide which was composed of 22 amino acids (ACHWPWCHGWHSACDLPMHPMC, abbreviated as sp22) and specifically bound to human CD59 was screened from a M13 phage display library so as to counteract tumor immune escape activity. The mechanism of exogenous sp22 peptide in inducing apoptosis of MCF‐7 cells was investigated. The results suggested that sp22 could lower CD59 expression level, downregulate Bcl‐2 expression, activate Fas and caspase‐3, and finally increase apoptotic cell numbers of MCF‐7 cells. However, sp22 had no obvious influence on normal human embryonic lung cells. In addition, the effects of endogenous sp22 gene on CD59 expression and NKM cell apoptosis were explored using the recombinant plasmid sp22‐PIRES. It showed that sp22 gene was efficiently expressed in transfected NKM cells. Compared with normal NKM cells, NKM cells transfected with sp22 displayed reduced mRNA and protein expression levels of CD59, increased sensitivity to complement‐mediated cytolysis, decreased cell survival ratio, changes of the expression of apoptosis associated proteins, increased number of apoptotic cells and the appearance of apoptotic morphology. The results suggested that sp22 protein could bind to CD59 and inhibit the expression of CD59. The cytolytic activity of complement on tumor cells strengthened and apoptosis signal was stepwise transferred which might be a potential way to kill tumor cells. J. Cell. Biochem. 113: 3810–3822, 2012. © 2012 Wiley Periodicals, Inc.     

Cell surface sialic acids do not affect primary CD22 interactions with CD45 and surface IgM nor the rate of constitutive CD22 endocytosis

CD22/Siglec-2 is a B cell-specific molecule modulating surface IgM (sIgM) signaling via cytosolic tyrosine-based motifs. CD22 recognizes alpha2-6-linked sialic acids (Sias) via an amino-terminal Ig-like domain. This Sia-binding site is typically masked by unknown sialylated ligands on the same cell surface, an interaction required for optimal signaling function. We studied the effect of cell surface Sias on specific interactions of CD22 with other molecules and on its turnover via endocytosis. A novel approach for simultaneous biotinylation and cross-linking showed that CD22 associates with CD45 and sIgM at much higher levels than reported in prior studies, possibly involving cell surface multimers of CD22. Sia removal or mutation of a CD22 arginine residue required for Sia recognition did not affect these associations even in human:mouse heterologous systems, indicating that they are primarily determined by evolutionarily conserved protein-protein interactions. Thus masking of the Sia-binding site of CD22 involves many cell surface sialoglycoproteins, without requiring specific ligand(s) and/or is mediated by secondary interactions with Sias on CD45 and sIgM. Abrogating Sia interactions also does not affect constitutive CD22 endocytosis. Sia removal does enhance the much faster rate of anti-CD22 antibody-triggered endocytosis, as well as killing by an anti-CD22 immunotoxin. In contrast to the unstimulated state, sIgM cross-linking inhibits both antibody-induced endocytosis and immunotoxin killing. Thus the signal- modulating activity of CD22 Sia recognition cannot be explained by mediation of primary interactions with specific molecules, nor by effects on constitutive endocytosis. The effects on antibody-mediated endocytosis could be of relevance to immunotoxin treatment of lymphomas.     

Ion binding of cyclolinopeptide A: an NMR and CD conformational study

CD and nmr techniques have been used to study, in acetonitrile solution, the ion-complexing capability of cyclolinopeptide A (CLA), a cyclic nonapeptide of sequence cyclo-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val) endowed with remarkable cytoprotective ability in vitro, and the conformation of the Ba(2+)/CLA complex. At room temperature, CLA in acetonitrile shows a proton nmr spectrum characteristic of the coexistence of many different conformers in intermediate exchange. The backbone contains a cis Pro-Pro bond, with all other peptide bonds in the trans conformation. CLA binds Ba2+ more tightly than the other cations studied, namely K+, Na+, Mg2+, and Ca2+; CD data are indicative of the presence of both 1:2 (sandwich) and 1:1 (equimolar) type complexes, depending on the Ba2+ ion concentration, whereas nmr data are consistent with an equimolar form. The relevant conformational features of the equimolar Ba2+/CLA complex are that the backbone contains all trans peptide bonds, a type I 6----3 beta-turn and a 3----1 gamma-turn (or a distorted 3----9 beta-turn). The global shape of the complexed peptide can be described as a bowl, with the concave (polar) side hosting Ba2+ and the convex side predominantly apolar.     

Identification of the gene variations in human CD22

 CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases. Received: 14 July 1998 / Revised: 7 September 1998     

Characterization of the nucleic acid binding region of adenovirus DNA binding protein by partial proteolysis and photochemical cross-linking.

The nucleic acid binding domain of the adenovirus type 2 (or type 5) DNA-binding protein (DBP) was characterized by using limited proteolysis and photochemical cross-linking. Three proteases were used to generate fragments of DBP which retained the ability to bind to single-stranded DNA. One fragment, a 35-kDa tryptic product, was partially sequenced and found to contain amino acid residues 153 to approximately 470. This fragment further defines the minimum region of the protein which is required for nucleic acid binding. The DNA binding pocket of DBP was defined by using ultraviolet irradiation to cross-link covalently the carboxyl-terminal portion of the protein to the oligonucleotide p(dT)14. Cross-linked complexes were digested with trypsin, and peptides which were associated with the oligonucleotide were isolated by anion-exchange and reverse-phase ion-pairing high performance liquid chromatography. Two DBP peptides comprised of residues 294-308 and 415-434 were isolated by this approach. Sequence analysis indicated that methionine 299 and phenylalanine 418 were probable sites of cross-linking between their respective peptides and the oligonucleotide; hence these residues may represent contact points between DBP and single-stranded DNA. Both residues are highly conserved and are near, but not identical to, regions of the protein implicated previously in DNA binding.     

A genomic snapshot of Salmonella enterica serovar Typhi in Colombia

Little is known about the genetic diversity of Salmonella enterica serovar Typhi (S. Typhi) circulating in Latin America. It has been observed that typhoid fever is still endemic in this part of the world; however, a lack of standardized blood culture surveillance across Latin American makes estimating the true disease burden problematic. The Colombian National Health Service established a surveillance system for tracking bacterial pathogens, including S. Typhi, in 2006. Here, we characterized 77 representative Colombian S. Typhi isolates collected between 1997 and 2018 using pulse field gel electrophoresis (PFGE; the accepted genotyping method in Latin America) and whole genome sequencing (WGS). We found that the main S. Typhi clades circulating in Colombia were clades 2.5 and 3.5. Notably, the sequenced S. Typhi isolates from Colombia were closely related in a global phylogeny. Consequently, these data suggest that these are endemic clades circulating in Colombia. We found that AMR in S. Typhi in Colombia was uncommon, with a small subset of organisms exhibiting mutations associated with reduced susceptibility to fluoroquinolones. This is the first time that S. Typhi isolated from Colombia have been characterized by WGS, and after comparing these data with those generated using PFGE, we conclude that PFGE is unsuitable for tracking S. Typhi clones and mapping transmission. The genetic diversity of pathogens such as S. Typhi is limited in Latin America and should be targeted for future surveillance studies incorporating WGS.     

Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: novel sialoside probe for murine CD22

The interaction of CD22 with glycoprotein ligands bearing the Siaalpha2,6Gal-R sequence is believed to modulate its function as a regulator of B cell signaling. Although a commercial sialoside-polyacrylamide (PAA) probe, NeuAc- alpha2,6Gal-PAA, has facilitated studies on ligand binding by human CD22, murine CD22 binds instead with high affinity to NeuGcalpha2,6Gal-R. A multivalent probe with this sequence was constructed to facilitate investigations of ligand binding in CD22 function using genetically defined murine models. The probe is based on the sialoside-PAA platform, which is then biotinylated for easy detection. A series of sialoside probes were constructed with two different length linker arms between the sialoside and the backbone and three different sialoside to PAA molar ratios. The NeuGcalpha2,6Gal-PAA probe is specific for CD22: it binds to sialidase-treated B cells of wild-type mice but not B cells of CD22-null mice. Additionally, because the probe only binds to sialidase-treated wild-type cells, it confirms that CD22 is constitutively "masked" on most B cells from wild-type mice by binding to ligands in cis. In contrast, the probe bound equally well to native or sialidase-treated B cells from the immunocompromised ligand-deficient ST6Gal I knockout mice, demonstrating that CD22 is constitutively "unmasked" in these cells.     

B cell antigen receptor and CD40 differentially regulate CD22 tyrosine phosphorylation

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at approximately 35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.