The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.     

Mutations in the MHC class I-like candidate gene for hemochromatosis in French patients

 A candidate gene for hemochromatosis has recently been localized on the short arm of chromosome 6, about 4 megabases telomeric to the major histocompatibility complex. It encodes a protein that exhibits significant similarity to the HLA class I molecules and can be provisionally designated HLA-hc. Genotype analysis of 94 hemochromatosis patients living in France and a similar number of controls confirms that the disease is strongly associated with homozygosity at nucleotide 845 (72% of the patients and none of the controls carry two copies of the 845A variant). The data are consistent with hemochromatosis being a heterogeneous disease: about 79% of the cases in this sample would be caused by a defect in HLA-hc and 21% by an unrelated mechanism. A second variant (187 G) enriched on patient chromosomes that do not carry the 845A mutation might influence the affinity of a ligand for HLA-hc; the exact nature of this ligand remains to be discovered. The 845A variant is the best genetic marker for the disease identified to date, and the detection of 845A homozygosity should now permit diagnosis of a readily curable disease and the prevention of sometimes deadly complications in at least 72% of the patients. Received: 30 September 1996 / Revised: 23 October 1996     

Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages

We have cloned human and murine DNase I-like cDNAs, termed LS-DNase, which are expressed at high levels in liver and spleen tissues. LS-DNase expression is highly specific to macrophage populations within these and other tissues. Mature LS-DNase from both species is a secreted, non-glycosylated protein containing 285 residues, with a calculated molecular mass of 33 kDa and a basic isolelectric point. Human and murine LS-DNase are highly conserved and share 83% identity. Sequence analysis reveals that LS-DNase shares 46% amino acid sequence identity with DNase I. However, several residues identified as important for interaction of human DNase I with actin are not conserved in both human and murine LS-DNase. Consistent with this observation, recombinant human LS-DNase possesses a DNA hydrolytic activity which, unlike DNase I, is not inhibited by G-actin. The existence of a family of DNase I-like molecules that have tissue-specific expression patterns and the possible role of a macrophage specific DNase are discussed.     

Physical assignment of the bovine MHC class IIa and class IIb genes.

Screening of a bovine yeast artificial chromosome (YAC) library revealed two clones which contain most of the class II genes of the major histocompatibility complex (MHC) known to date. The YACs were mapped by fluorescence in situ hybridization (FISH) and characterized for the class II genes they contain. We found that the classic class II genes BoLA- DQA, -DQB, -DRA, and -DRB3 are located at BTA 23q21 and the non-classic class II genes DYA, DIB, LMP2, LMP7, TAP2, BoLA-DOB, -DMA, -DMB, and -DNA are located at BTA 23q12-->q13. These two different mapping locations confirm and extend previous findings of a gross physical distance between classic and non-classic MHC class II genes in cattle.     

Molecular cloning of bovine class I MHC cDNA

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.     

NF-kappaB signaling regulates functional expression of the MHC class I-related neonatal Fc receptor for IgG via intronic binding sequences

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-alpha rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-kappaB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-kappaB (IkappaBalpha; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-kappaB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-kappaB binding sequences, respectively. The intronic NF-kappaB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-alpha stimulation or overexpression of NF-kappaB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-kappaB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-alpha stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-kappaB signaling via intronic sequences regulates FcRn expression and function.     

Cloning and characterization of receptor kinase class disease resistance gene candidates in Citrus

The rice gene Xa21 represents a unique class of plant disease resistance (R) genes with distinct protein structure and broad-spectrum specificity; few sequences or genes of this class have been cloned and characterized in other plant species. Degenerate primers were designed from the conserved motifs in the kinase domains of Xa21 and tomato Pto, and used in PCR amplification to identify this class of resistance gene candidate (RGC) sequences from citrus for future evaluation of possible association with citrus canker resistance. Twenty-nine RGC sequences highly similar to the kinase domain of Xa21 (55%–60% amino-acid identity) were cloned and characterized. To facilitate recovery of full-length gene structures and to overcome RGC mapping limitations, large-insert genomic clones (BACs) were identified, fingerprinted and assembled into contigs. Southern hybridization revealed the presence of 1–3 copies of receptor-like kinase sequences (i.e., clustering) in each BAC. Some of these sequences were sampled by PCR amplification and direct sequencing. Twenty-three sequences were thus obtained and classified into five groups and eight subgroups, which indicates the possibility of enhancing RGC sequence diversity from BACs. A primer-walking strategy was employed to derive full-length gene structures from two BAC clones; both sequences 17o6RLK and 26m19RLK contained all the features of the rice Xa21 protein, including a signal peptide, the same number of leucine-rich-repeats, and transmembrane and kinase domains. These results demonstrate that PCR amplification with appropriately designed degenerate primers is an efficient approach for cloning receptor-like kinase class RGCs. Utilization of BAC clones can facilitate this approach in multiple ways by improving sequence diversity, providing full-length genes, and assisting in understanding gene structures and distribution.Communicated by P. Langridge     

Molecular characterization of the porcine MHC class I region

A porcine cosmid library was screened with a human MHC class I cDNA. Four positive clones were isolated and mapped with different restriction endonucleases. Altogether nine SLA class I genes were identified and their positions located within restriction maps. Sizes of class I homologous DNA sequences varied between 3600 and 5800bp. The distances between these regions ranged from 11900 to 22200bp.     

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.     

MHC class I characterization of Indonesian cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) are quickly becoming a useful model for infectious disease and transplantation research. Even though cynomolgus macaques from different geographic regions are used for these studies, there has been limited characterization of full-length major histocompatibility complex (MHC) class I immunogenetics of distinct geographic populations. Here, we identified 48 MHC class I cDNA nucleotide sequences in eleven Indonesian cynomolgus macaques, including 41 novel Mafa-A and Mafa-B sequences. We found seven MHC class I sequences in Indonesian macaques that were identical to MHC class I sequences identified in Malaysian or Mauritian macaques. Sharing of nucleotide sequences between these geographically distinct populations is also consistent with the hypothesis that Indonesia was a source of the Mauritian macaque population. In addition, we found that the Indonesian cDNA sequence Mafa-B7601 is identical throughout its peptide binding domain to Mamu-B03, an allele that has been associated with control of Simian immunodeficiency virus (SIV) viremia in Indian rhesus macaques. Overall, a better understanding of the MHC class I alleles present in Indonesian cynomolgus macaques improves their value as a model for disease research, and it better defines the biogeography of cynomolgus macaques throughout Southeast Asia.     

The MHC class II-associated invariant chain interacts with the neonatal Fc gamma receptor and modulates its trafficking to endosomal/lysosomal compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.     

Cloning and characterization of a bovine adeno-associated virus

To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.     

Symmetry recognizing asymmetry: analysis of the interactions between the C-type lectin-like immunoreceptor NKG2D and MHC class I-like ligands

Engagement of diverse protein ligands (MIC-A/B, ULBP, Rae-1, or H60) by NKG2D immunoreceptors mediates elimination of tumorigenic or virally infected cells by natural killer and T cells. Three previous NKG2D-ligand complex structures show the homodimeric receptor interacting with the monomeric ligands in similar 2:1 complexes, with an equivalent surface on each NKG2D monomer binding intimately to a total of six distinct ligand surfaces. Here, the crystal structure of free human NKG2D and in silico and in vitro alanine-scanning mutagenesis analyses of the complex interfaces indicate that NKG2D recognition degeneracy is not explained by a classical induced-fit mechanism. Rather, the divergent ligands appear to utilize different strategies to interact with structurally conserved elements of the consensus NKG2D binding site.     

The nucleotide sequence of bovine MHC class IIDQB andDRB genes

The nucleotide sequences of most of the exons and parts of the introns of twoBoLA-DQB genes and twoBoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The twoDQB genes probably represent true alleles. Based on the exons sequenced, bothDQB genes and one of theDRB genes seem to be functional. The otherDRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occured in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease association in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30002–M30014. Address correspondence and offprint requests to: M. A. M. Groenen.     

Cloning and characterization of a cDNA encoding the beta-subunit of the bovine insulin-like growth factor-1 receptor.

This communication reports the sequence of the beta-subunit of the insulin-like growth factor-1 receptor. It shares 91.1% homology at the nucleotide level and 97.7% at the amino acid level with the equivalent human receptor subunit.     

Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16. To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M_r 44 000 and M_r 12000 polypeptides can be precipitated from both red and white cells isolated from M'-A16 positive animals, whereas no bands were seen in M'-A16 negative animals in precipitations with the same antibody. Precipitation with a crossreacting human β₂-microglobulin (β₂-m) specific antibody confirmed a class-I-like structure associated with β₂-m on M' positive red cells and the absence of such a structure on M' negative red cells. Sequential precipitations gave analogous results. Proteolytic degradation by papain and V8 protease did not reveal any substantial difference between red and white M'-A16 positive cells, but a slight difference in the pI of the immunoprecipitable components of red and white cells was observed. All together, this indicates that either the blood group antigen M' is the BoLA-A16 class I antigen or M' and BoLA-A16 are two different class I polypeptides with the same relative mass, sharing identical epitopes and both associated with β₂-m. Comparable results were obtained with M₁ and BoLA-A24.