Fucosylation of Cripto is required for its ability to facilitate nodal signaling.

O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors. A single CR point mutation, Thr-88 --> Ala, results in a form of the protein that is not fucosylated and has substantially weaker activity in cell-based CR/Nodal signaling assays, indicating that fucosylation is functionally important for CR to facilitate Nodal signaling.

An alternately spliced mRNA encoding functional domains of murine MAdCAM-1.

cDNA clones representing a small (0.8 kb) form of murine mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mRNA were obtained and sequenced. The sequence was identical to the published 1.6 kb murine MAdCAM-1 cDNA sequence, except that 432 nucleotides encoding the mucin-like and IgA-homologous portions were deleted. This cDNA most likely represents an alternately spliced mRNA. Substantial amounts of both the short and long MAdCAM-1 mRNAs are present in murine mesenteric lymph node. Ig fusion proteins displaying either the short or long forms of MAdCAM-1 can bind Mn(++)-activated JY cells bearing human alpha 4 beta 7 integrin, indicating that the two N-terminal Ig-like domains of MAdCAM-1 are sufficient to bind its integrin counter-receptor.

Molecular mapping of functional antibody binding sites of alpha 4 integrin.

Integrin alpha 4 beta 1 is a leukocyte receptor for fibronectin and vascular cell adhesion molecule-1 (VCAM-1). It is important in inflammatory recruitment of leukocytes, lymphopoiesis, and a number of development events. Here we have mapped a panel of functional monoclonal antibodies (mAbs) recognizing the integrin alpha 4 chain, using murine/human chimeric constructs expressed in COS7 cells. We find that: 1) mAbs that induce homotypic aggregation (epitope A mAbs) map to the most N-terminal 100 amino acids of the human alpha 4 chain; 2) mAbs that block adhesion of alpha 4 beta 1 to VCAM-1 and fibronectin (epitope B mAbs) map to a 52-amino-acid region between residues 152 and 203 of human alpha 4; 3) epitope B mAbs that do or do not induce aggregation (epitope B2 and B1 mAbs, respectively) map to the same regions and are therefore indistinguishable by this analysis; 4) mAbs that neither induce homotypic aggregation nor block adhesion (epitope C mAbs) map to a distinct region of the molecule comprising amino acids 422-606. The N-terminal region of the alpha 4 chain identified by functional A and B epitope mAbs does not correspond to ligand binding sites identified in other alpha subunits, such as cation binding sites or the "I-domain," which alpha 4 lacks, and thus represents a novel site for epitope functionality among the integrins.

Production and characterization of an analog of acidic fibroblast growth factor with enhanced stability and biological activity.

We have used recombinant DNA methods to produce two forms of bovine acidic fibroblast growth factor (aFGF), one with alanine substituted for the cysteine at position 47 and the other with the Ala47 change plus the substitution of glycine for the naturally occurring histidine at position 93. Both forms were expressed at high levels in Escherichia coli and purified to near homogeneity by solubilization of the inclusion bodies containing the aFGF, ion exchange chromatography, refolding of the protein and hydrophobic interaction chromatography. Circular dichroic and infrared spectra suggested that the proteins are similar in secondary and tertiary structures and contain little or no alpha-helical conformations. Hydrophobic interaction chromatography showed that aFGF C47A/H93G is slightly more hydrophobic than the aFGF C47A form, suggesting that residue 93 is exposed to the solvent. Half-maximal activity in an in vitro bioassay system was reached at a 10- to 20-fold lower dose for the aFGF C47A/H93G form than for the aFGF C47A form, suggesting that alteration of this residue has an effect on the region responsible for receptor binding. Addition of 50 micrograms/ml heparin enhanced the in vitro activity of the aFGFs, reducing the half-maximal dose to approximately 100 pg/ml for both forms, comparable to that observed previously for basic FGF with or without heparin in this assay system.

Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor.

Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.

Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine.

We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector. This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88. All three constructs were cloned and expressed in Escherichia coli and the proteins purified. The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF. The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays. Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups. This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond. Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration. Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation. These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.

Expression of hybrid class I genes of the major histocompatibility complex in mouse L cells.

The class I genes of the major histocompatibility complex of the mouse can be divided into two categories: those encoding the transplantation antigens and those encoding the Qa and Tla antigens. The inbred BALB/c mouse has 28 potential Qa/Tla genes. The sites of tissue expression, developmental regulation, and functions of these genes are virtually unknown. We have used the technique of exon shuffling to construct hybrid genes between each of three Qa region genes (Q5, Q7, and Q8) and two other class I genes (H-2Ld and Q6). The hybrid genes have been transfected into mouse L cells, in which intact transplantation antigen genes generally are expressed and in which intact Qa genes generally are not expressed. Analysis of expression of the hybrid gene constructs indicates that the 5' half of two of the Qa genes (Q5 and Q8) can readily be expressed in the context of a hybrid molecule, whereas the 3' half prevents cell-surface expression. The exon shuffling approach described here will be useful in characterizing Qa/Tla genes and in identifying or producing new reagents to study the Qa/Tla gene products, their tissue distribution, their developmental stages of expression, and, ultimately, their functions.

Expression and T cell recognition of hybrid antigens with amino-terminal domains encoded by Qa-2 region of major histocompatibility complex and carboxyl termini of transplantation antigens.

Coding potential of the Q6 gene from the Qa-2a region of BALB/c Crgl mice was analyzed by a combination of hybrid class I gene construction and DNA-mediated gene transfer. Recombinant genes were created by exon shuffling of the 5' coding region of the Q6 gene and the 3' coding region of a gene encoding a transplantation antigen (Kd, Dd, or Ld), or the inverse. Some of these hybrid class I genes were expressed in the transfected mouse fibroblasts (L cells). The hybrid class I molecules encoded by the 5' end of the Q6 gene and the 3' end of the Ld gene precipitated as 45,000 mol wt molecules associated with beta 2-microglobulin. The expression of the hybrid proteins indicates that 926 basepairs of the 5' flanking region upstream of the structural Q6 gene contain a promoter that functions as a transcription initiation site in L cells. The 3' portion of the Q6 gene appears to be responsible for the lack of cell surface expression of the intact Q6 and the hybrid Ld/Q6 genes in mouse fibroblasts. Accordingly, this portion of the Q6 class I gene may play a regulatory role in tissue-specific expression. Serological analyses of hybrid Q6 proteins suggested that Q6 may be a structural gene for CR (H-2 crossreactive) antigen found normally on subpopulations of lymphocytes. If this identification is correct, Q6 gene will define a new category of class I genes encoding approximately 40,000 mol wt molecules and carrying a characteristic truncated cytoplasmic tail. Analysis of L cells transfected with Q6 hybrid genes demonstrated also that the cytotoxic T cells specific for Qa-2a region-coded antigens recognize the amino-terminal alpha 1-alpha 2 domain of Q6 fusion products. This recognition can be blocked by anti-Qa-2a alloantiserum and monoclonal antibodies reactive with the alpha 3-beta 2-microglobulin portion of the Q6 hybrids. We propose that the structural requirements for the anti-Qa-2a cytotoxic T lymphocyte-specific epitopes on target molecules are the same as for anti-H-2-alloreactive cytotoxic T lymphocyte determinants on transplantation antigens and that the mechanism of target recognition is similar in both cases. This interpretation is consistent with the following structural similarities found in both categories of class I molecules: (a) Kd and Q6 alpha 1-alpha 2 domains share serologically defined epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic control of insulin receptors.

Insulin-binding activity was measured in hepatocyte suspensions and liver membrane preparations from newborn mice homozygous for a perinatal-lethal deletion at and around the albino locus in chromosome 7. Cell suspensions and membrane preparations from the mutant mice exhibited only 20-25% of the specific hormone-binding activity observed in comparable preparations from their homozygous normal and heterozygous littermates. The decrease in insulin-binding activity appears to be attributable to a decrease in the number of insulin receptor sites per cell rather than to a change in receptor affinity. Gene sequences deleted at and around the albino locus are therefore instrumental in the regulation of insulin receptor concentration rather than in coding for the insulin receptor itself. The results of the present studies extend the identification of the regulatory functions exerted by the genes around the albino locus of the mouse.