A New Apoprotein of Human Plasma Very Low Density Lipoproteins*

SUMMARY A new apoprotein has been isolated from delipidated human very low density lipoproteins by Sepharose 6B chromatography in 6 Y guanidine hydrochloride. This water-insoluble protein was noted to be unique to the plasma very low density and chylomicron fractions. This protein was shown to be homogeneous by urea-DEAE-cellulose chromatography, rechromatography in a guanidine-Sepharose system, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric focusing. The molecular weight of this apoprotein obtained by guanidine gel filtration, sedimentation equilibrium in guanidine hydrochloride, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 33,000. The amino acid content was significantly different from any previously characterized very low density lipoprotein apoprotein, containing relatively more arginine. By the dansyla-tion and cyanate techniques the NH2-terminal amino acid was found to be lysine. Digestion with carboxypeptidase revealed the COOH-terminal sequence to be -Leu-Ser-Ala-COOH.


SUMMARY
A new apoprotein has been isolated from delipidated human very low density lipoproteins by Sepharose 6B chromatography in 6 Y guanidine hydrochloride. This water-insoluble protein was noted to be unique to the plasma very low density and chylomicron fractions. This protein was shown to be homogeneous by urea-DEAE-cellulose chromatography, rechromatography in a guanidine-Sepharose system, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric focusing. The molecular weight of this apoprotein obtained by guanidine gel filtration, sedimentation equilibrium in guanidine hydrochloride, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 33,000. The amino acid content was significantly different from any previously characterized very low density lipoprotein apoprotein, containing relatively more arginine. By the dansylation and cyanate techniques the NH2-terminal amino acid was found to be lysine. Digestion with carboxypeptidase revealed the COOH-terminal sequence to be -Leu-Ser-Ala-COOH.
The major apoproteins of human plasma very low density lipoprotein consist of a group of low molecular weight proteins referred to as D proteins' (1,2), and a larger sized apo-low density lipoprotein (3). Each of these have been estimated to account for between 40 and 50% of the protein content of human VLDL2 (3,4). These previously characterized human VLDL proteins are not unique to this lipoprotein fraction and are isolated with other lipoproteins. The apo-LDL protein appears to be the predominant, if not the only protein of LDL (5), and the D proteins have been recovered in the human high density lipoprotein fraction (6 (24).

Sepharose Column
Chromatography-The results of Sepharose GB-guanidine hydrochloride column separation of reduced and alkylated apo-VLDL are shown in Fig. IA. Peak I appearing in the void volume of the 6B column was demonstrated by chromatography on the 4B system and by dodecyl sulfate polyacrylamide gel electrophoresis to be a protein with a molecular weight of about 300,000, approximately the same as that described by Smith et al. for apo-LDL (5). The protein was immunochemically identical to apo-LDL. Peak III was shown by similar techniques to be of the same molecular size as the D proteins described by Brown et al. (1, 2, 4), and reacted with antisera to apo-Glu and apo-Ala VLDL proteins.
Peak II had a molecular size unlike any previously described VLDL apoprotein. When Peak II fractions were concentrated and rechromatographed on the same system the protein was eluted in identical column fractions (Fig. 1B).

DEAE-cellulose
Chromatography-When total apo-VLDL was fractionated on the urea-DEAE cellulose system six apoprotein peaks were obtained. The column fractions were monitored at 280 nm and each peak was evaluated in a sodium dodecyl sulfate polyacrylamide gel electrophoresis system. The Peak II protein was eluted as a single peak, just after the second protein peak (apo-Glu) was eluted.
Polyacrylamide Gel Electrophoresis-The results of electrophoresis are shown in Fig. 2. The Peak II protein had a migration slightly greater than ovalbumin (mol wt 43,000) and slightly less than chymotrypsinogen (mol wt 27,000). Disulfide reduction did not change the migration of this protein, suggesting that no intra-or intermolecular disulfide bands were present. No carbohydrate could be detected in the Peak II band by periodic acid Schiff staining.
Gel Isoelectric Focusing-Isoelectric focusing runs of the Peak II protein indicate a single band with a p1 between 9 and 10 ( Fig. 3). Occasionally the 28,000 major apo-HDL protein was observed as a minor component on the isoelectric focusing runs. This amounted to a maximum of 5% of the total sample when evaluated by gel scanning densitometry.
Solubility Properties of Peak II Protein-Delipidated VLDL proteins were separately incubated at 37" on a mechanical shaker in 0.12 M Tris HCl, 0.03 M NaCl, pH 7.4, and 0.05 M phosphate, pH 7.0, buffers for 3 hours. The water-soluble peptides were determined in the supernatant after centrifuging the mixture at 6000 rpm for 20 min. The VLDL Peak II protein was not found among the aqueous soluble VLDL proteins in other than trace amounts by either the sodium dodecyl sulfate polyacrylamide gel electrophoresis system or by Sepharose-guanidine chromatography. Purified Peak II protein was found to be sparingly soluble in large volumes of aqueous buffer.
Amino Acid Analysis-The results of the amino acid analysis of 24-hour hydrolysates of protein from Peak II are shown in Table I. It will be noted that no cysteine is present. This is to be expected as a result of the reduction and alkylation procedure. However, carboxymethylcysteine was also not found, indicating that no cysteine was present in the native protein. The amino acid analyses were compared with those reported for other previously isolated and characterized components of VLDL (l-4). The new Peak II protein was distinctly different, particularly in its higher arginine content. It was similar to the other VLDL apoproteins in its low content of cysteine. The high arginine content of this protein is similar to that of DEAE Fractions 4, 5, and 6 of human delipidated VLDL described by Shore and Shore (26). Although other similarities between Peak II amino acid composition and that of Shore and Shore's proteins were observed, substantial differences were also noted.
Terminal Amino Acid Analysis-The only dansyl amino acid found after hydrolysis migrated with didansyl lysine in the twodimensional solvent systems (Fig. 4). The quantitative results of the Stark procedure also indicates lysine to be the NHz-termi- a Based on molecular weight = 33,000. Samples were obtained from two normals and two subjects with increased VLDL and hydrolyzed for 24 hours in 6 N HCl at 110". The maximal values for valine and isoleucine and the loss of threonine and serine were determined from more prolonged hydrolysis (see text). The data are presented f standard error of the mean.
b No carboxymethylcysteine was found in the reduced alkylated samples.
c This value is a mean of two individuals obtained by a method detailed in Ref. 14.

A B
FIG. 4. Chromatograms of dansyl amino acids. Plate A indicates reference dansyl amino acids which include proline, isoleucine didansyl lysine, glycine, glutamic acid, and serine (named in order from top to bottom and from lejt to right when in the same vertical dimension). Plate B represents the Peak II protein NH&erminus didansyl lysine and dansyl amine. Samples were spotted in the lower left hand corner and run in two dimensions as indicated in the text. nal amino acid (Table II). A value of 1.1 moles of lysine per mole of protein was obtained when the molecular weight was assumed to be 33,000.
Absence of Peak II Protein in Low and High Density Lipoproteins-Low density lipoproteins isolated between the density 1.020 and 1.063 demonstrated no protein of molecular size similar to Peak II protein when evaluated by either guanidine-Sepharose chromatography or sodium dodecyl sulfate polyacrylamide electrophoresis (Fig. 6). Traces of Peak II protein were, however, noted in the 1.006 to 1.019 low density fractions, especially in the patients with increased VLDL levels. No Peak II protein was ever observed, by the techniques indicated, for the high density lipoprotein fraction of normals or patients with increased VLDL.
The COOH-terminal amino acid of the Peak II protein was determined by digestion with carboxypeptidase, followed by amino acid analysis.
The results of these determinations are shown in Table III.
The results indicate that the COOH-terminal sequence is -Leu-Ser-Ala-COOH.
Most of the apoproteins of human VLDL that have already been isolated and defined have also been observed in other lipoprotein fractions of plasma.
The new protein which WC have identified appears to be unique to the triglyceride-rich lipoproteins of human plasma.
It is found predominantly in VLDL and in chylomicrons, and occasionally in trace amounts in the Molecular Weight Determinations-From the elution weights of reference proteins on the 6B Sepharose column a plot of Kd versus 8, 12 to 20 LDL fraction. ti Based on a molecular weight of 33,000 and corrected for blank runs.

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b Time of digestion at 37". log molecular weight was made. The elution weight of Peak II was determined. From this a mean value of 33,000 with a range of 30,000 to 36,000 was obtained for the molecular weight. When apo-VLDL was run on a Sepharose 4B column in the same solvent system a molecular weight of 39,000 was obtained for the protein.
The molecular weight of Peak II protein as determined from the mobility of this protein and reference proteins on a sodium dodecyl sulfate polyacrylamide gel electrophoresis system was also 33,000.
The molecular weight of Peak II was evaluated by sedimentation equilibrium in 6 M guanidine at rotor speeds of 20,000 and 22,000 rpm. Photoelectric scans were used to determine the absorbance versus distance from the axis of rotation.
A least squares program was used to calculate the slopes from the plots of In absorbance against r2 (Fig. 5). The partial specific volume of Peak II was calculated to be 0.74 from the amino acid composition and a mean molecular weight of 33,140 was determined. The protein was isolated from VLDL of both hyperlipoproteinemic and normal humans.
The molecular weight of 33,000 found for this new protein differs from the 250,000 obtained for the apo-LDL protein (5), 7,000 to 10,000 for the D proteins, and the 28,000 and 17,000 values obtained for the major HDL peptides (26). The uniqueness in size of this new lipoprotein apoprotein has aided its isolation and identification.
Although it has the same COOH-terminal amino acid as the apo-Ala protein of human VLDL, it has a different COOH-terminal sequence, a different NHrterminal amino acid, and a different amino acid content.
It is obviously not an aggregate of the 10,000 apo-Ala protein.
It does have a similarity to the amino acid composition of the D proteins in that both contain no cysteine or cystine residues. The large amount of arginine which this protein contains make its composition unique among human lipoproteins. The VLDL protein described by Shore and Shore (25) also has a high content of arginine and some similarity in amino acid content to the Peak II protein.
In the absence of further data characterizing their material it is impossible to decide if the proteins are in fact identical.
In contrast to the D proteins and the apo-LDL protein of VLDL, where the relative contents are functions of the size of the lipoprotein (27), this Peak II protein comprises from 5 to 15% of total VLDL protein for all sized lipoproteins.
It has been observed to be lost from VLDL when these lipoproteins are incubated with lecithin or lecithin-cholesterol mesophase dispersions and is then recovered in the LDL flotation region along with the mesophase lipid.
It is also found in substantial concentrations in the LDL fraction of humans with cholestatic liver disease who have significant amounts of LDL lecithin and cholesterol in mesophase form (28). The role which this protein plays in the metabolism of the VLDL lipid is currently under study.
FIG. 6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the apoproteins from the major lipoprotein classes. Solvent was 0.05 M phosphate buffer, pH 7.0, containing 0.1% sodium dodecyl sulfate. The numbering of VLDL proteins is the same as in Fig. 2.