Generation of a monoclonal antibody that recognizes a polymorphic epitope of C57BL/6 Ly-49G2.

To facilitate the study of natural killer (NK) cell functions, we have generated a panel of hybridomas that secrete antibodies recognizing B6 NK cell surface markers. One of these hybridomas, Cwy-3, secretes a mouse IgG1 specific for the B6 allele of Ly-49G2 (Ly-49G2B6). In addition, this monoclonal antibody (MAb) fails to stain A-LAKs derived from all other mouse strains tested. This suggests that Cwy-3 recognizes a polymorphic epitope of Ly-49G2B6. More importantly, this MAb exhibits no cross-reactivity to other known B6 Ly-49 family members. Therefore, Cwy-3 is in sharp contrast to the two known anti-Ly-49G2 MAbs, which are reported to recognize a nonpolymorphic epitope of Ly-49G2, and react with other Ly-49 members. In this regard, Cwy-3 will offer significant advantages over the cross-reactive anti-Ly-49G2 MAbs in fully defining the specificity and function of the Ly-49G2B6 NK cell receptor. With this novel Ly-49G2-specific MAb, we have isolated an EL-4 subline expressing a significant density of Ly-49G2 receptors.

Specificity and function of activating Ly-49 receptors.

Inhibitory Ly-49 receptors allow murine natural killer (NK) cells to kill cells with aberrant class I MHC expression while sparing normal cells. This is accomplished by their recognition of specific class I MHC products and prevention of NK-cell lysis of cells that present a normal repertoire of class I MHC ligands--"the missing self hypothesis". However, Ly-49 receptors that lack the cytoplasmic immunoreceptor tyrosine-based inhibitory motif, which is required for inhibition of killing, have also been described. These receptors were found to stimulate NK killing and are therefore referred to as activating Ly-49 receptors. Interestingly, the activating receptors have class I MHC-binding domains that are nearly indistinguishable from those of the inhibiting receptors, and binding to class I MHC has now been demonstrated for three activating receptors. Presently, there is no defined physiological role for activating Ly-49 receptors. Here we present an overview of current knowledge regarding the diversity, structure and function of activating Ly-49 receptors with a focus on class I MHC specificity, and we discuss their potential role(s) in natural resistance.

Lipid transfer particle-induced transformation of human high density lipoprotein into apolipoprotein A-I-deficient low density particles.

Incubation of human high density lipoprotein (HDL) particles (density = 1.063-1.21 g/ml) with catalytic amounts of Manduca sexta lipid transfer particle (LTP) resulted in alteration of the density distribution of HDL protein such that the original HDL particles were transformed into new particles with an equilibrium density = 1.05 g/ml. Concomitantly, substantial amounts of protein were recovered in the bottom fraction of the density gradient. The LTP-induced alteration in HDL protein density distribution was dependent on the LTP concentration and incubation time. Electrophoretic analysis revealed that the lower density fraction contained apolipoprotein A-II (apoA-II) as the major apoprotein component while nearly all of the apoA-I was recovered in the bottom fraction. Lipid analysis of the HDL substrate and product fractions revealed that the apoA-I-rich fraction was nearly devoid of lipid (less than 1%, w/w). The lipid originally associated with HDL was recovered in the low density, apoA-II-rich, lipoprotein fraction, and the ratios of individual lipid classes were the same as in control HDL. Electron microscopy and gel permeation chromatography experiments revealed that the LTP-induced product lipoprotein population comprised particles of larger size (19.7 +/- 1.4-nm diameter) than control HDL (10.6 +/- 1.4-nm diameter). The results suggest that facilitated net lipid transfer between HDL particles altered the distribution of lipid such that apoprotein migration occurred and donor particles disintegrated. Similar results were obtained when human HDL3 or HDL2 density subclasses were employed as substrates for LTP. The lower surface area to core volume ratio of the larger, product lipoprotein particles compared with the substrate HDL requires that there be a decrease in the total exposed lipid/water interface which requires stabilization by apolipoprotein. Selective displacement of apoA-I by apoA-II or apoC, due to their greater surface binding affinity, dictates that apoA-I is preferentially lost from the lipoprotein surface and is therefore recovered as lipid-free apoprotein. Thus, it is conceivable that the structural arrangement of HDL particle lipid and apoprotein components isolated from human plasma may not represent the most thermodynamically stable arrangement of lipid and protein.

Ly-49W, an activating receptor of nonobese diabetic mice with close homology to the inhibitory receptor Ly-49G, recognizes H-2D(k) and H-2D(d).

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2(k) or H-2(d) haplotypes, and Ab-blocking experiments indicated that H-2D(k) and D(d) are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2(k) class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.

A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s.

The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains coexist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ly-49CB6 NK inhibitory receptor recognizes peptide-receptive H-2Kb.

NK-mediated cytotoxicity involves two families of receptors: activating receptors that trigger lysis of the target cells being recognized and inhibitory receptors specific primarily for MHC I on the target cell surface that can override the activating signal. MHC I molecules on the cell surface can be classified into molecules made stable by the binding of peptide with high affinity or unstable molecules potentially capable of binding high affinity peptide (hence, peptide receptive) and being converted into stable molecules. It has been previously shown that the Ly-49A inhibitory receptor recognizes stable Dd molecules. We show in this study that the inhibitory receptor Ly-49CB6 recognizes peptide-receptive Kb molecules, but does not recognize Kb molecules once they have bound high affinity peptide.

Ly-49P activates NK-mediated lysis by recognizing H-2Dd.

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd alpha1/alpha2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.