The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.

In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq^® Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70–100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.

Selectively labeled polypeptides comprising the gamma-carboxyglutamic acid (Gla) domain (GD) and helical stack (HS) regions of human protein C (PC), and consisting of amino acid residues 1-47, have been chemically synthesized and their Ca2+ binding properties assessed by [13C]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [13C] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [13C] chemical shifts, titrations of which were used to obtain apparent dissociation constants for each Gla residue in the presence of all other such residues. The Ca2+ titration data obtained on each of the nine polypeptides showed that Gla residues 6, 16, 25, and 26 were involved in the higher affinity Ca2+ binding sites, whereas the remaining Gla residues, viz., 7, 14, 19, 20, and 29, coordinated Ca2+ more weakly. The results are consistent with conclusions drawn from functional studies obtained with site-directed mutations of individual Gla residues and with the structural model of the GD/HS of human PC. In these cases, Gla residues 6, 16, and 26 served as coordination loci for internally located Ca2+ ions, and GD-related Ca(2+)- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.

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We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.

Background: Conventional methods for conjugation of macromolecules, such as antibodies and reporter groups, typically yield a mixture ranging from unconjugated starting materials to large aggregates. We explored the use of a solid-phase process to allow improved control in conjugation of macromolecules for use in immunodiagnostic reagents. Methods: Activated components were sequentially delivered to an immobilized core protein, linking in concentric layers. For immunodiagnostic reagents, proteins with the desired signaling properties were added as interior layers and binding proteins were placed in the final surface layer. After assembly, the conjugates were released into solution by cleaving the linker holding the core protein to the support. Conjugates were prepared with use of three different reporter agents: R-phycoerythrin for microsphere fluorescence flow immunoassay, alkaline phosphatase for enzyme immunoassay, and acridinium for magnetic chemiluminescence immunoassay. For each reporter, six conjugates were prepared with various concentrations of both the reporter and an antibody directed against the ␣-subunit of thyroidstimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH. Results: Products ranged in mass from ϳ1 to ϳ20 MDa. HPLC analysis of the conjugates on a gel-permeation column showed sizes and chromophore contents highly consistent with the intended structures. In appropriate assay formats, the signal generated by a conjugate increased with incubation time, then plateaued at an intensity approximately proportional to the reporter content but relatively independent of the antibody con-

The Ca' + binding properties of various y-carboxyglutamic acid (g1a)-containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca2 + -selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca2 , with some peptides probably involved in intermolecular bridging of the Ca2 + .The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g-atom of Ca2 + interacts with 2 mol of peptide ( n = 0.5) with a Kd value of 1.6 mM. A second g-atom of Ca2 + interacts with 2 mol of this same peptide (n = 0.5) and is characterized by a K d of 8.8 mM. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (17 = 2.0) for Ca2 + ofKd = 16.1 mM, as well as a tighter site ( n = l), Of Kd = 0.4 mM. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38-residue peptide that possesses all nine of the gla-residues of protein C in their proper sequence positions. The strongest Ca2 + binding sites ( n = 3-4) possess an average Kd of 0.3 mM, followed by another class of sites (n = 5-10, average Kd = 1.5-3.0 mM). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca2 + to the gla region of prothrombin fragment 1. By selective [ 13C]-labeling of the essential gla 16 residue of the 38-mer peptide, we demonstrate that this particular gla residue participates as a donor for a high-affinity Ca2 + site.These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca' + to intact gla-containing proteins.Calcium is a required component of the blood coagulation cascade, and functions through its interaction with several proteins, viz. the vitamin K-dependent proteins, that participate in this pathway. Important to Ca2 + binding to these proteins, which include prothrombin, factors VII, IX and X, as well as proteins C and S, is the post-translational modification of specific precursor E residues to gla residues. This latter series of reactions employs vitamin K as a cofactor for the functional y-carboxylase, and results in formation of 9-13 residues of gla, the first 10 of which are present Abbreviations: gla, y-carboxyglutamic acid ; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high-performance liquid chromatography: hPC, human protein C; Ot-Bu, terr-butyl ester; 0-Bzl, benzyl ester; t-Boc, terr-butyloxycarbonyl; Pmc, 2,2,5,7,8-pentamethyIchroman-6-sulfonyl; Bum, terr-butyloxymethyl; Z , benzyloxycarbonyl; FAB, fast atom bombardment.

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