Reduced fetal hepatic alpha-fetoprotein levels in Down's syndrome.

In the majority of pregnancies involving a Down's syndrome (DS) fetus, the level of alpha-fetoprotein (AFP) measured in maternal serum and amniotic fluid is reduced to about 70 per cent of the level attained in normal pregnancies. Causes of this decrease may include the production of an altered AFP molecule with modified turnover or transport properties, or a reduction in the level of AFP synthesis. We examined hepatic AFP mRNA transcripts and compared AFP polypeptide isoforms in liver tissue samples obtained from a group of DS and normal abortuses. No differences was detected in the structure of the AFP mRNA transcript or in the charge or mass of AFP polypeptides in the two sample groups. However, the hepatic AFP level, expressed as microgram AFP/mg protein, was significantly lower in a group of 28 DS cases relative to a group of 47 normal controls (p = 0.04). This difference in hepatic AFP concentration did not appear to be the result of a general reduction in the level of total protein or total RNA production. The greatest difference between the AFP levels of the DS and normal groups was observed in the earliest samples examined (i.e., at 17-19 weeks of age) where the median AFP levels differed by about 20 per cent.

Expression of myelin proteins in the developing human spinal cord: cloning and sequencing of human proteolipid protein cDNA.

A full-length clone for the human proteolipid protein (PLP) was isolated from a cDNA library constructed from poly(A)+ RNA isolated from fetal spinal cords obtained at 15-24 weeks of conceptional age. The sequence of the human PLP cDNA was determined, and the deduced amino acid sequence was found to be identical with that of rat PLP. Comparison of human and rat PLP cDNA clones indicated that the coding regions retained 97% homology and that there were also other areas of conserved sequence. The human 5'-untranslated region was 93% homologous to that of the rat. The 3'-untranslated region was, overall, 73% homologous to that of the rat with areas containing greater than 84% homology in the first 400 and last 200 nucleotides. The most variability within the 3'-untranslated region occurred between nucleotides 2,000-2,500, where homology with the rat cDNA dropped to 55%. Expression of PLP in the human spinal cord between 11 and 23 weeks after conception was examined and compared with the expression of the myelin basic protein (MBP). RNA was isolated from pooled human spinal cords obtained at three periods of development: 11-14 weeks, 17-19 weeks, and 21-23 weeks. Northern blot analysis revealed a 3.2-kilobase (kb) PLP mRNA that was present at higher abundance in the 21-23-week spinal cord RNA than in the 17-19-week or the 11-14-week samples. The 17-19-week RNA sample also contained a PLP-hybridizing band at 2.2 kb which may possibly have arisen by utilization of alternative polyadenylation signals. Messenger RNA for MBP was detectable at 11-14 weeks but was readily evident in both the 17-19- and 21-23-week age groups. Immunoblot analysis of whole spinal cord homogenates indicated that polypeptides for MBP preceded the appearance polypeptides for PLP by 3-4 weeks.

Evidence for the expression of four myelin basic protein variants in the developing human spinal cord through cDNA cloning.

Four human myelin basic protein (MBP) variants with molecular masses of 21.5, 20.2, 18.5, and 17.3 kilodaltons (kDa) have been identified in the developing human spinal cord and their structures determined through an analysis of cDNA clones of their mRNAs. The 20.2-kDa MBP mRNA encoded a novel MBP variant, the structure of which has not been reported in any species. Its amino acid sequence was identical with that of the 21.5-kDa MBP except for a deletion of 11 amino acid residues encoded by exon 5 of the MBP gene. All four human MBP variants were identical except for the insertion of deletion of two peptide fragments corresponding to those encoded by exons 2 and 5 of the MBP gene. In this study, no mature human MBP cDNAs missing exon 6 sequences were identified. This suggests that, unlike the mouse, the four human MBP mRNAs encoding these MBP variants arise by the alternative splicing of only exons 2 and 5 from the primary MBP gene transcript. This indicates that the predominant MBP splicing pathways in human and mouse are different. Immunoblots of human fetal spinal cords (11-21 weeks) indicated that MBP expression turned on abruptly between 14 and 16 weeks. Expression of the 20.2-kDa MBP variant was most evident at 16 weeks and its relative proportion declined thereafter, suggesting that its expression was developmentally regulated.

Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis.

Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Several non-coat polypeptides appeared to segregate exclusively with different populations as resolved by two-dimensional electrophoresis. Stripped CVs also exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetylcholine esterase, and an Mr 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.

Detection of novel fetal polypeptides in human amniotic fluid using two-dimensional gel electrophoresis.

We compared the two-dimensional polypeptide pattern of donor-matched samples of mid-gestational amniotic fluid and maternal plasma to identify fetally-derived polypeptides in the amniotic fluid. Ten major polypeptide groups, ranging in molecular mass from 14.6 to 225 Kilodaltons (K), were consistently present in amniotic fluid that were not detected in maternal plasma. Three of these polypeptide complexes have been identified: one is alphafetoprotein, the second is a cellular form of fibronectin, and the third is beta 2-microglobulin. We searched for the remaining seven polypeptides in fetal serum and urine to confirm their fetal origin. Urine contained two polypeptides at 30.5K and 25.5K with mobilities identical to species found in amniotic fluid; the 25.5K polypeptide was also present in cord serum. Possible sources for the remaining 5 components may be the fetal respiratory and gastrointestinal tracts, integument, membranes (amnion and chorion) or the uterus. In addition, developmental changes in amniotic fluid polypeptides were studied by comparing samples obtained at mid-gestation and at term. Three proteins characteristic of mid-gestational amniotic fluid with masses of 185.8K, 38.2K and 25.4K were not detected or were present only at trace levels at term; and new polypeptide groups at approximately 51K, and 18K were observed in late gestation.

Characterization of fetal urinary proteins at midgestation and term.

Proteins in first voided urine from premature and term neonates were analyzed by two-dimensional gel electrophoresis. At 22-28 weeks of gestation, urine contained many identifiable serum proteins and the presence of an array of minor polypeptides with masses below 40 kilodaltons (kd) made the patterns quite complex. In contrast, the abundance of serum proteins in term urine was markedly decreased and the polypeptide patterns approached the relative simplicity seen in adult urine. Urine contained three major nonserum polypeptide groups at 30.5, 33-49, and 97 kd. The 97-kd species was identified as the Tamm-Horsfall glycoprotein, which is known to be derived from the renal tubule.

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins.

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.

Enzymatic conversion of proteins to glycoproteins by lipid-linked saccharides: a study of potential exogenous acceptor proteins.

Previous studies have shown that a membrane preparation from hen oviduct catalyzes transfer of oligosaccharide from oligosaccharide-P-P-dolichol to denatured RNase and alpha-lactalbumin. To gain further insight into the structural requirements of a protein that allow it to serve as a substrate for glycosylation, the acceptor ability of a variety of other modified proteins containing the tripeptide sequence-ASN-X-(SER/THR)-has been investigated. Of 7 proteins tested, 2 (ovine prolactin and rabbit muscle triosephosphate isomerase) could be enzymatically glycosylated by a particulate preparation from hen oviduct. The remaining 5 proteins, assayed as either S-carboxymethylated or S-aminoethylated derivatives, were inactive as carbohydrate acceptors. However, cyanogen bromide treatment of 2 of the inactive proteins, bovine catalase and concanavalin A from jack bean, yielded peptide fragments which served as substrates for glycosylation. These results suggests that for some proteins, disruption of the tertiary structure is sufficient to allow attachment of carbohydrate. Other denatured proteins may possess additional restrictions imposed by their secondary structure. In certain cases, these restrictions are removed when the polypeptide chain is fragmented.