Corrective effect of a single intravenous injection of mouse LAMAN

As shown in section 3.1.2, the mouse LAMAN had a higher content of Man6P-recognition marker (73.6%) as compared to LAMAN of the other two species.

Due to the high content of M6P groups, a higher uptake of the enzyme by the lysosomes was expected. Therefore mouse LAMAN was selected for the first replacement therapy with α-mannosidosis knock-out mice.

In order to study the short and the long term effect of a single dose of LAMAN on the storage of neutral oligosaccharides, eight α-mannosidosis mice at the age of 9 weeks received 100 mU mouse LAMAN per gram body weight (3.6 µl/g body weight) intravenously (2.2.4.2), and the mice were killed 2 h to 16 days after injection (2.2.4.3). Mouse LAMAN pattern (see section 3.1.1), was composed of about 90% precursor and 10% proteolytically processed forms.

One α-mannosidosis mouse was injected with PBS and used like mock-control.

Analysis of alpha-mannosidase activity (2.2.2.10) in the serum (2.2.2.4) of blood taken 5 minutes after injection revealed that in the mice killed after 8 h and 1 day the greater part of the enzyme had been injected paravenously. The analysis was therefore restricted to mice killed after 2 h, 4 h, 10 h, 4 days, 8 days and 16 days.

3.2.1 Clearance in serum

In order to control for the amount of injected enzyme and its clearance from the circulation blood was taken from the retroorbital plexus 5, 30 and 60 minutes after injection (2.2.4.2) and LAMAN activity was determined (2.2.2.10). Five min after injection the LAMAN activity varied by less than 6% indicating that the mice had received comparable amounts of enzyme. The enzyme was cleared

from circulation with a half life of 19.4 minutes and a standard deviation of 1.5 minutes (Table 3.2).

LAMAN activity in serum (mU/ml) Mouse

killed after (h)

5 min 30 min 60 min

2h 1928 616 65 4h 1849 827 303 10h 2068 840 414

Table 3.2: LAMAN activity in serum of α-mannosidosis mice after injection of 100 mU mouse LAMAN per g body weight.

3.2.2 Stability and distribution of the enzyme

Mouse LAMAN injected α-mannosidosis mice were anaesthetized and killed by intracardial perfusion with PBS 2, 4 and 10 h after injection (2.2.4.3) and organ extracts were prepared for determination of LAMAN activity (2.2.2.10). Uptake of mouse LAMAN was seen into all organs except brain.

The maximum activity of LAMAN in the organs was observed 2-4 h after injection (Table 3.3). In liver the activity was 6-7 times higher than in control mice, but also in spleen and heart the maximum values exceeded those of controls. In kidney at maximum one fourth of the activity in control mice was reached. The small activity seen in brain may be due to enzyme located in the vascular system. The data from table 3.3 demonstrate that the LAMAN activity persists in brain at a constant level for at least 10 hours. As the LAMAN activity in the blood rapidly decreases with time, this observation suggests that the LAMAN activity in brain extract is not blood derived.

LAMAN (mU/g wet weight)

Liver Spleen Kidney Heart Brain

(+/+)*

Table 3.3: LAMAN activity in tissue extracts of control and α-mannosidosis mice before and after injection of 100 mU mouse LAMAN per g body weight.

* +/+ refers to control mice, -/- to α-mannosidosis mice, and n to the number of animals investigated.

+ All values were corrected for the mean α-mannosidase activity in serum at tzero (2350 mU/ml serum). The correction factors were 0,98, 1,07 and 0,96 for 2, 4 and 10h respectively.

Between 4 and 10 h after injection the enzyme activity decreased rapidly in liver, spleen and kidney with an apparent half life of 2.8, 2.6 and 2.9 hours respectively. The immunoblot showed in Fig 3.2 demonstrates that the internalised precursor of LAMAN (130 kDa) was rapidly processed in liver to mature forms. 2 and 4 hours post injection only proteolytically processed forms of ~70 kDa and ~35 kDa (not seen in mock injected mouse) were detectable.

Fig. 3.2: Western Blot (WB) of mouse liver.

20 µg proteins from mouse liver were separated by 10% SDS-PAGE (2.2.2.6) and transferred to PVDF membranes (2.2.2.7).

Rabbit antiserum raised against recombinant human LAMAN, (1:50000) was used as primary antibody and horseradish peroxidase (HRP) (1:20000) as coupled secondary antibody.

→ Endogenous alpha-mannosidase

* Non specific signals

3.2.3 Short effect of the treatment

Organ extracts were also prepared for the determination of neutral oligosaccharides (2.2.3.1). The hallmark of α-mannosidosis is the storage of neutral oligosaccharides in a wide variety of tissues (see Fig. 3.3, lane 1 and lane 2; Stinchi et al., 1999)). As determined by mass spectrometry (2.2.3.6) and sensitivity to jack bean α-mannosidase (2.2.2.4, see outcome in Fig 3.10), the oligosaccharides M2 to M9 contain 2 to 9 mannose residues and a single N-acetylglucosamine residue at the reducing terminus, and therefore result from the action of an endoglucosaminidase. The amount of neutral oligosaccharides quantified by densitometry (2.2.3.3) in liver and spleen decreased progressively with time to 15% and to 7% of that observed in mock-injected animals, while in kidney and heart the storage was reduced only to about 49% and 74% (Fig.

3.3).

Fig. 3.3: Thin-layer chromatography of neutral oligosaccharides in liver, spleen, kidney and heart.

The fraction of neutral oligosaccharides from equal amounts of tissues was separated by thin-layer chromatography (2.2.3.2).

Lane 1 contains the sample from mock-injected control mice (+/+), lane 2 from mock-injected α-mannosidosis mice (-/-), lane 3, 4 and 5 the samples from α-mannosidosis mice injected with 100 mU mouse LAMAN per g body weight and killed after 2, 4 and 10 h, respectively (see also Table 3.3). Oligosaccharides were detected with orcinol/ sulphuric acid (2.2.3.2) and quantified by densitometry (2.2.3.3). For calculation of the storage the amount of neutral oligosaccharides in α-mannosidosis mice was corrected for that in mock-injected control mice. The storage in LAMAN injected α-mannosidosis mice was expressed as percentage of that in mock-injected α-mannosidosis mice.

3.2.4 Duration of the treatment effect

To determine how long the corrective effect of a single dose of mouse LAMAN persisted, three α-mannosidosis mice were examined 4, 8 and 16 days after the injection of 100 mU mouse LAMAN per g body weight. At all time points the LAMAN activity in the organs (spleen, kidney, heart, brain) was in the range of non-treated or mock-injected α-mannosidosis mice (see table 3.3), except for liver, where 4 days after injection the LAMAN activity (15.1 mU/g wet weight) was still about 5-fold higher than in mock-injected mice.

Fig. 3.4: Neutral oligosaccharides in tissue extracts of α-mannosidosis mice injected with 100 mU mouse LAMAN per g body weight.

The mice were killed 4, 8 and 16 days after injection and the neutral oligosaccharides in extracts of liver, spleen, kidney and heart were separated by TLC (2.2.3.2) and quantified by densitometry (2.2.3.3).

The storage of neutral oligosaccharides (as calculated in section 3.2.3) in liver, spleen and kidney 4 days after injection was in the range observed 10 h after injection (compare Fig. 3.3 and Fig. 3.4). In heart storage had decreased from 74% after 10 h to 42% 4 days after injection. This indicates that the corrective effect seen after 10 h persisted for about 4 days in spite of the fact that little or no LAMAN activity is detectable 10 h after injection in organs such as kidney or heart. After 4 days the storage of oligosaccharides clearly began to increase again. The increase observed between day 4 and day 16 after injection corresponded to 20-40% of the storage seen in mock-injected α-mannosidosis mice (Fig. 3.4).

3.3 Comparison of the clearance and corrective effect of