PETER G. H. BYFIELD

Abetalipoproteinemia is an inherited disorder of lipoprotein metabolism. Affected individuals produce virtually no circulating apolipoprotein B-containing lipoproteins (chylomicrons, very low density lipoprotein, low density lipoprotein and lipoprotein (a)). Malabsorption of the antioxidant vitamin E occurs, leading to spinocerebellar and retinal degeneration. Biochemical and genetic studies show that abetalipoproteinemia is not a defect of lipid biosynthesis or of the apolipoprotein B gene. Instead a microsomal triglyceride transfer protein, which exists as a complex with protein disulphide isomerase in the endoplasmic reticulum, has been implicated. We have cloned and sequenced the human cDNA encoding microsomal triglyceride transfer protein. The predicted amino acid sequence shows extensive homology to vitellogenin, the precursor of the lipovitellin complex, which has been shown by X-ray crystallography to contain a large lipid storage cavity. Microsomal triglyceride transfer protein is expressed in ovary, testis and kidney, in addition to liver and small intestine. A homozygous mutation that disrupts splicing has been identified in affected siblings with classical abetalipoproteinemia. These results elucidate a key process in the packaging of apolipoprotein B with lipid, and should increase our understanding of the processes regulating the production of atherogenic lipoproteins.

IAPP, or amylin, is a 37-amino acid peptide that is co-secreted with insulin from the pancreatic beta-cells. We have determined the effects of IAPP and the antagonist 8-37 fragment of IAPP on the secretion of insulin from isolated rat islets studied in a perifusion system. Insulin secretion was stimulated by 8 mM glucose and 0.2 microM carbachol. IAPP at 10(-7) M reduced insulin release by 32% from 7.1 (95% Cl 5.8-8.6) to 4.8 (3.0-7.5) fmol.min-1 x islet-1 (P = 0.046, n = 7). IAPP at 1.5 x 10(-6) M reduced insulin release by 62% from 6.5 (3.4-12.3) to 2.5 (1.4-4.4) fmol.min-1 x islet-1 (P = 0.001, n = 6). IAPP at 10(-5) M decreased insulin release by 70% (P < 0.001, n = 6). When IAPP (8-37) at 10(-5) M was added to IAPP at 1.5 x 10(-6) M, there was only a 22% reduction of insulin release (P = 0.06, n = 6) compared with control chambers with no peptide added. This reduction was less (P = 0.002) than observed with IAPP (1.5 x 10(-6) M) alone. IAPP (8-37) at 4 x 10(-5) M in the absence of exogenously added IAPP increased insulin secretion by 48% (P = 0.01, n = 6), but IAPP (8-37) at 10(-5) M did not alter insulin secretion. These findings demonstrate that IAPP decreases insulin secretion from islet beta-cells, an effect that can be antagonized by the 8-37 fragment of IAPP.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic peptide [TAP25, (T1aAPPAHGVT9S10APDT14RPAPGS20)T1bAPPA5b] corresponding to one repeat (T1a-S20) and five overlapping amino acids (T1b-A5b) of the MUC1 core protein served as an acceptor substrate for in vitro glycosylation. TAP25 was glycosylated using the detergent-solubilized UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases from the breast carcinoma cell line T47D, the colon carcinoma cell line HT29 and from human premature skim milk. The glycosylated peptides were isolated by ultrafiltration, purified by reverse-phase HPLC and further analysed by liquid secondary ion mass spectrometry (LSIMS). Three different glycosylation species, mono-, di- and triglycosylated peptides were identified. Automated Edman sequencing and LSIMS of proteolytic fragments independently revealed the sites of GalNAc incorporation and confirmed that the threonine residues Thr9 and Thr1b are the preferred sites of glycosylation independent of the enzyme source, while Thr14 remained non-glycosylated even with the enzyme preparation from milk. In addition, evidence was obtained that at least 20% of the glycosylated peptides exhibited GalNAc incorporation at Ser20. On the basis of kinetic studies a preferred sequence of GalNAc addition to the three acceptor sites has been concluded (Thr9-->Thr1b-->Ser20). Although Thr14 within the PDTRP motif of the tandem repeats remained non-glycosylated, the introduction of GalNAc into adjacent positions significantly decreased the immunoreactivity of antibodies SM-3, HMFG-1 and HMFG-2 defining overlapping epitopes of this motif. It is assumed that glycosylation at Thr9, Thr1b and Ser20 distorts the peptide conformation of the binding epitope.

Factor VIII (FVIII) is the nonproteolytic cofactor for FIXa in the tenase complex of blood coagulation. FVIII is proteolytically activated by thrombin and FXa in vitro to form a heterotrimer with full procoagulant activity. Activated protein C inactivates thrombin-activated FVIII through cleavage adjacent to position Arg 336 in the cofactor. We have investigated the interaction of FIXa and FVIII and subjected FVIII polypeptides to N-terminal amino acid sequence analysis. Contrary to previous reports, we were unable to demonstrate the activation of FVIII by FIXa. Incubation of these two proteins at equimolar or close to equimolar concentrations resulted in the inactivation of FVIII, coincident with cleavage of the FVIII heavy chain adjacent to Arg 336 and the light chain adjacent to Arg 1719. These cleavages were detected in the presence or absence of thrombin, indicating that FIXa does not stabilize thrombin-activated FVIIIa. APC cleaved FVIII at the same position in the heavy chain, and simultaneous incubation of FVIII, APC, and FIXa did not result in stabilization of the cofactor. We conclude that FIXa does not play a role in the stabilization or activation of FVIII.