Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.

Human mammary cells present on the cell surface a polymorphic epithelial mucin (PEM) which is developmentally regulated and aberrantly expressed in tumors. PEM carries tumor-associated epitopes recognized by the monoclonal antibodies HMFG-1, HMFG-2, and SM-3. Previously isolated partial cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 20-amino acid repeat units. We now report the full sequence for PEM, as deduced from cDNA sequences. The encoded protein consists of three distinct regions: the amino terminus consisting of a putative signal peptide and degenerate repeats; the major portion of the protein which is the tandem repeat region; the carboxyl terminus consisting of degenerate tandem repeats and a unique sequence containing a transmembrane sequence and a cytoplasmic tail. Potential O-glycosylation sites (serines or threonines) make up more than one-fourth of the amino acids. Length variations in the tandem repeat result in PEM being an expressed variable number tandem repeat locus. Tandem repeats appear to be a general characteristic of mucin core proteins.     

Comparative structure and evolution of murine CR2. The homolog of the human C3d/EBV receptor (CD21)

The complete nucleotide sequence of murine complement receptor type 2 (CR2) was determined from two overlapping cDNA clones derived from a lambda gt11 library of late pre-B cell origin. Comparison of the predicted sequence of the 1014 amino acid murine homolog with that of human CR2 revealed marked evolutionary conservation. The murine molecule was 65% identical to human CR2 overall, lacking a single repetitive sequence variably present in man. The 15 approximately 60-75 amino acid short consensus repeats (SCR) that constitute the entire extracellular domain of murine CR2 were 53 to 81% identical to and could be directly aligned with the human protein. As reported, the cytoplasmic tail shared 79% amino acid identity with human CR2, whereas that of the transmembrane was only 33%. Murine CR2 contained 16 potential N-linked glycosylation sites of which 6 were conserved, 4 altered, and 6 lost during human evolution. The hydropathicity profile of the two molecules was nearly colinear with some variation in the N-terminal region of the first repeat, as well as within the sixth and twelfth repeats. RNA blot analysis revealed a approximately 4.0 to 5.0 kb message in murine B lymphocytes, which was absent in T lymphocytes (thymus and spleen), liver, brain, lung, kidney, and heart. A method was devised to more precisely compare the repeat structures. An identity matrix analysis suggests that human ancestral CR2 evolved before divergence of the rodent and primate branches of the evolutionary tree through a series of predictable gene duplications, possibly giving rise to the precursor of human CR1 and murine CRY. The marked structural similarity between the human and murine receptors suggests functional conservation as well.     

Expansion of the complement receptor gene family. Identification in the mouse of two new genes related to the CR1 and CR2 gene family

Human cDNA probes encoding the C3b/C4b complement receptor, CR1, have been used to identify, in the mouse, two new genes which are related to CR1 but which appear to encode a different protein product. These new mouse genes, arbitrarily designated mouse genes X and Y, hybridize specifically to three different cDNA probes derived from human CR1. The degree of hybridization homology between the mouse X and Y genes suggests they are very closely related to one another; however, the chromosomal localization of the mouse X gene to chromosome 8 and the mouse Y gene to chromosome 1 indicates they are distinct gene sequences. The mRNA species detected with the X and/or Y (X/Y) sequences are approximately 2000 bases in length, but vary in both quantity and size depending upon the tissue analyzed. DNA sequence analysis of a cDNA specific for the X and Y sequences indicates the mature protein(s) will contain the 60 amino acid consensus repeat characteristic of a group of other proteins including CR1, the C3d receptor (CR2), H, C4 binding protein (C4bp), the interleukin 2 (Il 2) receptor and others. The identity of the mouse X and Y genes, and the function of the proteins which they encode, is not known; however, the small size of the mRNA and the tissue specific expression suggests they do not encode mouse CR1 or CR2 but instead encode a related protein (or proteins) which is expressed in a wide variety of mouse tissues.     

Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

Abstract The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum , was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M _r value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of M _r 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.     

Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction.

The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7.